ES2975235T3 - Hydantoins that modulate BACE-mediated APP processing - Google Patents

Abstract

In certain embodiments, provided herein are hydantoin compounds that are effective at inhibiting BACE activity against APP. Without being bound by a particular theory, it is believed that the activity of the hydantoins identified herein appears to be associated with binding to BACE and/or APP particularly when these moieties form a BACE/APP complex. Accordingly, the compounds described herein are believed to represent a new class of compounds designated herein as APP-binding BACE inhibitors (ABBIs) and provide a novel mechanism for modulating APP processing. The hydantoins described herein appear to exhibit enhanced brain permeability and functional inhibition of BACE.

Description

DESCRIPTION

Hydantoins that modulate BACE-mediated APP processing

Cross reference to related requests

This application claims the benefit and priority of USSN 61/763.830, filed February 12, 2013.

STATEMENT OF GOVERNMENTAL SUPPORT

[Not applicable]

Background

Amyloid beta peptide (Ap) is a primary component of amyloid beta fibrils and plaques, which are considered to play a role in an increasing number of pathologies. Examples of such conditions include, but are not limited to, Alzheimer's disease, Down syndrome, Parkinson's disease, memory loss (including memory loss associated with Alzheimer's disease and Parkinson's disease), attention deficit symptoms (including attention deficit symptoms associated with Alzheimer's disease, Parkinson's disease and Down syndrome), dementia (including presenile dementia, senile dementia, dementia associated with Alzheimer's disease, Parkinson's disease and Down syndrome), progressive supranuclear palsy, cortical basal degeneration, neurodegeneration, olfactory impairment (including olfactory impairment associated with Alzheimer's disease, Parkinson's disease and Down syndrome), p-amyloid angiopathy (including cerebral amyloid angiopathy), hereditary cerebral hemorrhage, mild cognitive impairment ("MCI"), glaucoma, amyloidosis, type II diabetes, hemodialysis (p2 microglobulins and complications thereof), neurodegenerative diseases such as sheep scrapie, spongiform encephalitis bovine, Creutzfeld Jakob disease, traumatic brain injury and the like.

Ap peptides are short peptides that are produced by proteolysis of the transmembrane protein called amyloid precursor protein ("APP"). Ap peptides are obtained from the cleavage of APP by p-secretase activity at a position near the N-terminus of Ap, and by gamma secretase activity at a position near the C-terminus of Ap. (APP is also cleaved by α-secretase activity, resulting in the non-amyloidogenic secreted fragment known as soluble APPa.) The beta-site APP-cleaving enzyme ("BACE-1") is considered the major aspartyl protease responsible for the production of Ap by p-secretase activity. Inhibition of BACE-1 has been shown to inhibit Ap production.

Alzheimer's disease (AD) is estimated to affect more than 20 million people worldwide and is thought to be the most common cause of dementia. As the global population ages, the number of people with Alzheimer's disease (AD, currently approximately 5.4 million in the United States), will continue to increase. Alzheimer's disease is a neurodegenerative disease associated with progressive dementia and memory loss. Two key features of AD are accumulation of extracellular deposits containing aggregated Ap peptide and neuronal synaptic loss in AD in specific brain regions. Although the pathogenesis of AD is complex, compelling genetic and biochemical evidence suggests that Ap overproduction or failure to clear this peptide is the first event in the amyloid cascade leading to AD primarily through amyloid deposition, which is hypothesized to be involved in neurofibrillary tangle formation, neuronal dysfunction, and microglia activation, which characterize brain tissues affected by AD.

Ap accumulation is considered the first event in a complex cascade leading to neurodegeneration, as demonstrated by compelling genetic and biochemical evidence. The amyloid cascade hypothesis (Hardy and Allsop (1991) Trends Pharmacol. Sci.,12: 383-388; Selkoe (1996) J. Biol. Chem.,271: 18295-18298; Hardy (1997) Trends Neurosci.,20: 154-159; Hardy and Selkoe (2002) Science,297: 353-356) states that overproduction of Ap, or the inability to eliminate this peptide, leads to AD, mainly through amyloid deposition, which is assumed to participate in the formation of neurofibrillary tangles, neuronal dysfunction and microglial activation, which are characteristics of brain tissues affected by AD (Busciglio et al. (1995) Neuron,14: 879-888; Gotzet al.(1995)EMBO J,14: 1304-1313; Lewisetal.(2001)Science,293: 1487-1491; Hardyet al.(1985)Nat Neurosci.,1: 355-358).

Considering the causal role of Ap in the etiology of AD, novel therapeutic strategies that reduce Ap levels or prevent the formation of neurotoxic Ap species have been suggested as a method to prevent or delay disease progression. Indeed, the main focus during the last decade has been to inhibit brain Ap production and aggregation, increase parenchymal Ap clearance, and interfere with Ap-induced cell death.

Sequential cleavage of APP by the membrane-bound proteases p-secretase and Y-secretase results in the formation of Ap. A competing proteolytic route to the p-secretase pathway, the a-secretase pathway, results in cleavage of APP within the Ap domain, preventing the generation of Ap (Selkoe(2001) Physiol. Rev.,81: 741-766; Hussainet al.(1999)Mol. Cell. Neurosci.,14: 419-427; Sinhaet al.(1999)Nature,402: 537-540; Vassaret al.(1999)Science,286: 735-741). The p-site APP cleavage enzyme 1 (BACE1) was identified as the major p-secretase activity mediating the first cleavage of APP in the p-amyloidogenic pathway(Id.).

BACE1 is a 501 amino acid protein that has homology to eukaryotic aspartic proteases, especially of the pepsin family (Yanet al. (1999) Nature,402:533-537). Like other aspartic proteases, BACE1 is synthesized as a zymogen with a prodomain that is cleaved by furin to release the mature protein. BACE1 is a type I transmembrane protein with a luminal active site that cleaves APP to release an ectodomain (sAPPp) into the extracellular space. The remaining C-terminal fragment (CTF) undergoes further cleavage by Y-secretase, leading to the release of Ap and the APP intracellular C-terminal domain (AICD).

Presenilins have been proposed to be the major enzymatic component of Y-secretase, whose imprecise cleavage of APP produces a spectrum of Ap peptides varying in length by a few amino acids at the C-terminus. Most Ap normally terminate at amino acid 40 (Ap40), but the 42-amino acid variant (Ap42) has been shown to be more susceptible to aggregation, and is hypothesized to nucleate senile plaque formation. Modulation of Y-secretase can also lead to an increase in the 38-amino acid variant (Ap38). The competitive α-secretase pathway results from sequential cleavages by α-secretase and Y-secretase. Three metalloproteases from the disintegrin and metalloprotease family (ADAM 9, 10, and 17) have been proposed as candidates for the α-secretase activity, which cleaves APP at position 16 in the Ap sequence. Using overexpression experiments, ADAM-10 has been shown to be the likely α-secretase for APP cleavage (Vassar (2002) Adv. Drug Deliv. Rev.,54:1589-1602; Buxbaume et al. (1998) J. Biol. Chem,273:27765-27767; Koike et al.(1999)Biochem. J ,343(Pt 2):371-375). This cleavage also releases an ectodomain (sAPPa), which shows neuroprotective functions (Lammichet al. (1999) Proc. Natl. Acad. Sci. USA,96: 3922-3927). Subsequent cleavage of the 83 amino acid CTF (C83) releases p3, which is non-amyloidogenic, and AICD (Furukawa et al. (1996) J. Neurochem,67: 1882-1896). The functions of these fragments are not completely clear, although AICD is hypothesized to mediate intracellular signaling.

Research elucidating the metabolic pathways that regulate Ap production from amyloid precursor protein (APP) indicates that Ap-producing secretases are good therapeutic targets, since inhibition of either p-secretase or Y-secretase limits Ap production. The fact that p-secretase initiates APP processing and thus serves as the rate-limiting step in Ap production, its inhibition has attracted efforts from many research groups. Examples from the patent literature are growing and include, for example, WO2006009653, W02007005404, WO2007005366, WO2007038271 WO2007016012, US2005/0282826, US2007072925, WO2007149033, WO2007145568, WO2007145569, WO2007145570, WO2007145571, WO2007114771, US20070299087, WO2005/016876, WO2005/014540, WO2005/058311, WO2006/065277, WO2006/014762, WO2006/014944, WO2006/138195, WO2006/138264, WO2006/138192, WO2006/138217, WO2007/050721, WO2007/053506, 25, WO2006/138230, WO2006/138265, WO2006/138266, WO2007/053506, WO2007/146225, WO2008/073365, WO2008/073370, WO2008/103351, US2009/041 201, US2009/041202 and WO2010/047372.

A limitation of protease inhibitor strategies is the inhibition of cleavage of all substrates of a given targeted protease, such as BACE or the Y-secretase complex. In the case of Y-secretase, non-APP substrates, such as Notch, raise concerns about potential side effects of Y-secretase inhibition and the recent failure of the Y-secretase inhibitor. Problems associated with the use of semagacestat serve to reinforce such concerns.

BACE is a key enzyme involved in APP processing leading to Ap42 production and Alzheimer's disease (AD) pathology. BACE-1 (also called BACE) has become a popular area of research since its discovery, and has perhaps surpassed Y-secretase as the most promising target for pharmaceutical research. One problem with Y-secretase as a target is its known cleavage of Notch, which serves important functions in neuronal development. Presenilin knockout mice demonstrated abnormal somitogenesis and axial skeletal development with shortened body length as well as cerebral hemorrhages (Shenet al.(1997)Cell,89:629-639; Wonget al.(1997)Nature,387:288-292). In contrast, several groups have reported that BACE1 knockout mice are healthy and show no signs of adverse effects (Luo et al. (2001) Nat. Neurosci.,4: 231-232; Roberd et al. (2001) Hum. Mol. Genet.,10: 1317-1324), while one group noted subtle neurochemical deficits and behavioral changes in fertile and viable mice (Harrison et al. (2003) Mol. Cell Neurosci.,24: 646-655). Although recent studies have shown that BACE1 knockout mice exhibit hypomyelination of peripheral nerves (Willemet al. (2006) Science,314: 664-666), the consequences of BACE1 inhibition in adult animals, where myelination has already taken place, are unclear. BACE1 has recently been reported to cleave multiple substrates, including ST6Gal I, PSGL-1, voltage-gated sodium channel subunits, APP-like proteins (APLP), LDL receptor-related protein (LRP), and most recently neuregulin 1 type III (NRG1) (Willemet al.(2006) Science,314:664-666; Huet al. (2006) Nat. Neurosci.,9:1520-1525). Therefore, the consequences of directly inhibiting BACE1 are not yet fully understood.

Molecular modeling (Sauderet al.(2000)J. Mol. Biol.,300:241-248) and subsequent X-ray crystallography (Honget al.(2000)Science,290:150-153; Maillardet al.(2007)J. Med. Chem.,50:776-781) of the BACE-1 active site in complex with a transition state inhibitor provided crucial insights into BACE-1-substrate interactions. Structurally, the BACE-1 active site is more open and less hydrophobic than other aspartyl proteases, making the development of effective BACE inhibitor candidates in vivo difficult. While there is a large drug discovery effort focused on the development of direct BACE inhibitors, none have so far significantly advanced into clinical testing.

Some BACE inhibitors such as LY2811376 and CTS21166 entered clinical trials but did not advance beyond Phase 1 due to safety concerns. The discovery of other physiological substrates of BACE poses a major concern in the clinical development of BACE inhibitors or BACE modulators and could be a significant obstacle in the advancement of these inhibitors as a therapy for the disease.

Summary

The present invention is defined in the appended claims. Embodiments not within the scope of the appended claims do not form part of the invention. Any reference in the description to methods of treatment refers to the compounds, pharmaceutical compositions and medicaments of the present invention for use in a method for the treatment of the human (or animal) organism by therapy. In certain embodiments, provided herein are hydantoin compounds that are effective in inhibiting BACE activity against APP. Without intending to be bound by a particular theory, it is believed that the activity of the hydantoins identified herein appears to be associated with binding to BACE and/or APP, particularly when these moieties form a BACE/APP complex. Accordingly, it is believed that the compounds described herein represent a new class of compounds designated herein as APP-binding BACE inhibitors (ABBIs) and provide a new mechanism for modulating APP processing. The hydantoins described herein appear to show improved brain permeability and functional inhibition of BACE.

The present invention provides a selection of compounds as set forth in the claims and their uses in therapy. In addition, for informational purposes only, examples of other compounds not considered to be within the scope of the claims are also disclosed herein.

Definitions

Unless otherwise indicated, reference to a compound (e.g., to a hydantoin as described herein) should be construed broadly to include pharmaceutically acceptable salts, tautomers, alternative solid forms, non-covalent complexes, and combinations thereof, of a chemical entity of the depicted structure or chemical name.

Generally, reference to a particular element, such as hydrogen or H, is intended to include all isotopes of that element. For example, if a group R is defined to include hydrogen or H, it also includes deuterium and tritium. Accordingly, isotopically labeled compounds are within the scope of this invention.

A pharmaceutically acceptable salt is any salt of the parent compound that is suitable for administration to an animal or human. A pharmaceutically acceptable salt also refers to any salt that can be formed in vivo as a result of administration of an acid, another salt. A salt comprises one or more ionic forms of the compound, such as a conjugate acid or base, associated with one or more corresponding counterions. Salts may be formed by or incorporate one or more deprotonated acidic groups (e.g., carboxylic acids), one or more protonated basic groups (e.g., amines), or both (e.g., zwitterions).

A prodrug is a compound that is converted to a therapeutically active compound after administration. For example, the conversion may occur by hydrolysis of an ester group, such as a C<1>-C<6> alkyl ester of the carboxylic acid group of the present compounds, or some other biologically labile group. The preparation of prodrugs is well known in the art. For example, "Prodrugs and Drug Delivery Systems", which is a chapter in Richard B. Silverman, Organic Chemistry of Drug Design and Drug Action, 2nd Ed., Elsevier Academic Press: Amsterdam, 2004, pp. 496-557, provides further details on the subject.

Tautomers are isomers that are in equilibrium with each other. For example, tautomers can be related by the transfer of a proton, a hydrogen atom, or a hydride ion.

Unless stereochemistry is explicitly described, a structure is intended to include all possible stereoisomers, both pure and in any possible mixtures.

Alternative solid forms are solid forms other than those that may result from practicing the methods described herein. For example, alternative solid forms may be polymorphs, different types of amorphous solid forms, glasses, and the like. In various embodiments, alternative solid forms of any of the compounds described herein are contemplated.

Generally, "substituted" refers to an organic group as defined below (e.g., an alkyl group) in which one or more bonds to a hydrogen atom contained therein are replaced by a bond to atoms other than hydrogen or carbon. Substituted groups also include groups in which one or more bonds to one or more carbon or hydrogen atoms are replaced by one or more bonds, including double or triple bonds, to a heteroatom. Thus, a substituted group may be substituted by one or more substituents, unless otherwise specified. In some embodiments, a substituted group is substituted by 1, 2, 3, 4, 5, or 6 substituents. Examples of substituent groups include: halogens (i.e., F, Cl, Br, and I); hydroxyls; alkoxy, alkenoxy, alkynoxy, aryloxy, aralkyloxy, heterocyclyloxy, and heterocyclylalkoxy groups; carbonyls (oxo); carboxyls; esters; urethanes; oximes; hydroxylamines; alkoxyamines; aralkoxyamines; thiols; sulfides; sulfoxides; sulfones; sulfonyls; sulfonamides; amines; N-oxides; hydrazines; hydrazides; hydrazones; azides; amides; ureas; amidines; guanidines; enamines; imides; isocyanates; isothiocyanates; cyanates; thiocyanates; imines; nitro groups; nitriles (i.e., CN), and the like.

The term "alkyl" refers to and includes any and all groups that are known as normal alkyl, branched chain alkyl, cycloalkyl, and also cycloalkyl-alkyl. Illustrative alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, octyl, and decyl. The term "cycloalkyl" refers to cyclic, including cyclic, saturated hydrocarbyl groups. Examples include, but are not limited to, cyclopentyl, cyclohexyl, dicyclopentyl, norbornyl, octahydronaphthyl, and spiro[3.4]octyl. In certain embodiments, alkyl groups contain 1-12 carbon atoms (C<1>-<12> alkyl), or 1-9 carbon atoms (C<1>-<9> alkyl), or 1-6 carbon atoms (C<1>-<6> alkyl), or 1-5 carbon atoms (C<1>-<5> alkyl), or carbon atoms (C<1>-<4> alkyl), or 1-3 carbon atoms (C<1>-<3> alkyl), or 1-2 carbon atoms (C<1>-<2> alkyl).

By way of example, the term "C<1>-<6> alkyl group" refers to a straight or branched chain alkyl group having 1 to 6 carbon atoms, and can be illustrated by a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a ferc-butyl group, a sec-butyl group, an n-pentyl group, a ferc-amyl group, a 3-methylbutyl group, a neopentyl group and an n-hexyl group.

As used herein, the term "alkoxy" means an alkyl group attached through a single terminal oxygen atom. An "alkoxy" group may be represented as --O-alkyl, where alkyl is as defined above. The term "aryloxy" is used similarly, and may be represented as --O-aryl, with aryl as defined below. The term "hydroxy" refers to --OH.

Similarly, the term "alkylthio" as used herein means an alkyl group attached through a single terminal sulfur atom. An "alkylthio" group may be represented as —S-alkyl, where alkyl is as defined above. The term "arylthio" is used similarly, and may be represented as --S-aryl, with aryl as defined below. The term "mercapto" refers to --SH.

Aryl groups are cyclic aromatic hydrocarbons that contain no heteroatoms. Aryl groups include monocyclic, bicyclic, and polycyclic ring systems. Thus, aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenylenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenyl, anthracenyl, indenyl, indanyl, pentalenyl, and naphthyl groups. In some embodiments, aryl groups contain 6-14 carbons, and in others 6 to 12 or even 6-10 carbon atoms in the ring portions of the groups. Although the term "aryl groups" includes groups containing fused rings, such as fused aromatic-aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl, and the like), it does not include aryl groups having other groups, such as alkyl or halo groups, appended to one of the ring members. In contrast, groups such as tolyl are referred to as substituted aryl groups. Representative substituted aryl groups may be monosubstituted or substituted more than once. For example, monosubstituted aryl groups include, but are not limited to, 2-, 3-, 4-, 5-, or 6-substituted phenyl or naphthyl groups, which may be substituted with substituents such as those listed above.

The term "heteroaryl group" refers to a monocyclic or fused ring aromatic heterocyclic group containing one or more heteroatoms selected from O, S and N. If the aromatic heterocyclic group has a fused ring, it may include a partially hydrogenated monocyclic group. Examples of such a heteroaryl group include a pyrazolyl group, a thiazolyl group, an isothiazolyl group, a thiadiazolyl group, an imidazolyl group, a furyl group, a thienyl group, an oxazolyl group, an isoxazolyl group, a pyrrolyl group, an imidazolyl group, a (1,2,3)- and (1,2,4)-triazolyl group, a tetrazolyl group, a pyranyl group, a pyri group. dil, a pyrimidinyl group, a pyrazinyl group, a pyridazinyl group, a quinolyl group, an isoquinolyl group, a benzofuranyl group, an isobenzofuranyl group, an indolyl group, an isoindolyl group, an indazolyl group, a benzoimidazolyl group, a benzotriazolyl group, a benzoxazolyl group, a benzothiazolyl group, a benzo[ b]thiophenyl, a thieno[2,3-b]thiophenyl group, a (1,2) and (1,3)-benzoxathiol group, a chromenyl group, a 2-oxochromenyl group, a benzothiadiazolyl group, a quinolizinyl group, a phthalazinyl group, a naphthyridinyl group, a quinoxalinyl group, a quinazolinyl group, a cinnolinyl group and a carbazolyl group.

A "derivative" of a compound means a chemically modified compound where the chemical modification takes place at one or more functional groups of the compound. However, the derivative is expected to retain or enhance the pharmacological activity of the parent compound.

As used herein, "administer" refers to local and systemic administration, for example, including enteral, parenteral, pulmonary, and topical/transdermal administration. Routes of administration for the agents (e.g., the hydantoins described herein, or one or more tautomers or stereoisomers thereof, or pharmaceutically acceptable salts or solvates of said one or more hydantoins, said one or more stereoisomers, or said one or more tautomers thereof) that find use in the methods described herein include, for example, oral administration (per os (p.o.)), nasal or inhalation administration, administration as a suppository, topical contact, transdermal delivery (e.g., via a transdermal patch), intrathecal (IT) administration, intravenous ("iv") administration, intraperitoneal ("ip") administration, intramuscular ("im") administration, intralesional administration, or subcutaneous ("sc") administration, or implantation of a slow release device, e.g., an osmotic minipump, a depot formulation, etc., to a subject. Administration may be by any route, including parenteral and transmucosal (e.g., oral, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular, ionophoretic, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.

The terms "systemic administration" and "systemically administered" refer to a method of administering the one or more agents described herein or the composition to a mammal so that the one or more agents or the composition are delivered to sites in the body, including the target site of pharmaceutical action, via the circulatory system. Systemic administration includes, but is not limited to, oral, intranasal, rectal, and parenteral administration (e.g., other than via the alimentary tract, such as intramuscular, intravenous, intraarterial, transdermal, and subcutaneous).

The term "co-administer" or "concurrent administration" or "administer together with" when used, for example, with respect to the one or more active agents described herein, e.g., the hydantoins described herein, or one or more tautomers or stereoisomers thereof, or pharmaceutically acceptable salts or solvates of said one or more hydantoins, said one or more stereoisomers, or said one or more tautomers thereof and a second active agent (e.g., a cognition enhancer), refers to the administration of the one or more agents and the second active agent such that both can simultaneously achieve a physiological effect. The two agents, however, need not be administered together. In certain embodiments, the administration of one agent may precede that of the other. The simultaneous physiological effect does not necessarily require the presence of both agents in the circulation at the same time. However, in certain embodiments, co-administration typically results in both agents being present simultaneously in the body (e.g., in plasma) at a significant fraction (e.g., 20% or more, preferably 30% or 40% or more, more preferably 50% or 60% or more, most preferably 70% or 80% or 90% or more) of their peak serum concentration for any given dose.

The term "effective amount" or "pharmaceutically effective amount" refers to the amount and/or dosage and/or dosing regimen of one or more agents necessary to achieve a desired result, e.g., an amount sufficient to mitigate in a mammal one or more symptoms associated with mild cognitive impairment (MCI), or an amount sufficient to lessen the severity or delay the progression of a disease characterized by amyloid deposits in the brain in a mammal (e.g., therapeutically effective amounts), an amount sufficient to reduce the risk of, or delay the onset of, and/or reduce the ultimate severity of a disease characterized by amyloid deposits in the brain in a mammal (e.g., prophylactically effective amounts).

The term "causing to be administered" refers to actions taken by a medical professional (e.g., a physician), or a person who controls the medical care of a subject, that control and/or permit the administration of the one or more agents in question to the subject. Causing to be administered may involve diagnosis and/or determination of an appropriate therapeutic or prophylactic regimen, and/or prescribing the particular one or more agents for a subject. Such prescribing may include, for example, writing a prescription form, taking a medical history, and the like.

As used herein, the terms "treat" and "treatment" refer to delaying the onset of, slowing or reversing the progress of, reducing the severity of, or alleviating or preventing the disease or condition to which the term applies or one or more symptoms of such disease or condition.

The term "mitigate" refers to the reduction or elimination of one or more symptoms of that pathology or disease, and/or a reduction in the rate or delay of the onset or severity of one or more symptoms of that pathology or disease, and/or the prevention of that pathology or disease. In certain embodiments, reducing or eliminating one or more symptoms of pathology or disease may include, but is not limited to, reducing or eliminating one or more markers that are characteristic of the pathology or disease (e.g., total Tau (tTau), phospho-Tau (pTau), APPneo, soluble Ap40, pTau/Ap42 ratio, and tTau/Ap42 ratio, and/or increasing in the CSF the levels of one or more components selected from the group consisting of Ap42/Ap40 ratio, Ap42/Ap38 ratio, sAPPa, sAPPa/sAPPp ratio, sAPPa/Ap40 ratio, sAPPa/Ap42 ratio, etc.) and/or reducing, stabilizing, or reversing one or more diagnostic criteria (e.g., clinical dementia rating (CDR)).

As used herein, the term "consisting essentially of" refers to the genera or species of active pharmaceutical agents recited in a method or composition and may further include other agents that, by themselves, do not have substantial activity for the recited indication or purpose. In some embodiments, the term "consisting essentially of" expressly excludes the inclusion of one or more additional agents having neuropharmacological activity other than the one or more recited agents (e.g., other than ASBIs such as galangin and rutin). In some embodiments, the term "consisting essentially of" expressly excludes the inclusion of one or more additional active agents other than the one or more active agents described herein (e.g., other than ASBIs such as galangin and rutin). In some embodiments, the term "consisting essentially of" expressly excludes the inclusion of one or more acetylcholinesterase inhibitors.

The terms "subject," "individual," and "patient" refer interchangeably to a mammal, preferably a human or non-human primate, but also to domesticated mammals (e.g., canine or feline), laboratory mammals (e.g., a mouse, rat, rabbit, hamster, guinea pig), and agricultural mammals (e.g., an equine, bovine, porcine, ovine). In various embodiments, the subject may be a human (e.g., an adult male, an adult female, an adolescent male, an adolescent female, a boy, a girl) under the care of a physician or other healthcare worker in a hospital, psychiatric care facility, as an outpatient, or other clinical setting. In certain embodiments, the subject may not be under the care or prescription of a physician or other healthcare worker.

As used herein, the term "formulation" or "drug formulation" or "dosage form" or "pharmaceutical formulation" refers to a composition containing at least one therapeutic agent or medicament for delivery to a subject. In certain embodiments, the dosage form comprises a particular "formulation" or "drug formulation" and may be administered to a patient in the form of a lozenge, pill, tablet, capsule, suppository, membrane, strip, liquid, patch, film, gel, aerosol, or other form.

The term "mucous membrane" generally refers to any of the mucus-coated biological membranes of the body. In certain embodiments, the one or more active agents described herein can be administered through any mucous membrane found in the body, including, but not limited to, the buccal, perlingual, nasal, sublingual, pulmonary, rectal, and vaginal mucosa. Of interest is absorption through the mucous membranes of the oral cavity and the gut. Thus, peroral, buccal, sublingual, gingival, and palatal absorption are contemplated herein.

The term "transmucosal" delivery of a drug and the like is intended to encompass all modes of delivery across a mucous membrane.

As used herein, the term "bioadhesion" refers to the process of adhesion of one or more dosage forms to a biological surface, e.g., mucous membranes.

"Controlled drug delivery" refers to the release or administration of a drug from a given dosage form in a controlled manner to achieve the desired pharmacokinetic profile in vivo. One aspect of "controlled" drug delivery is the ability to manipulate the formulation and/or dosage form to establish the desired drug release kinetics.

"Sustained drug delivery" refers to the release or administration of a drug from a source (e.g., a drug formulation) in a sustained manner over an extended but specific period of time, which may extend from several minutes to a few hours, days, weeks, or months. In various embodiments, the term "sustained" will be used to refer to the delivery of constant and/or substantially constant levels of drug over a period of time ranging from a few minutes to a day, with a profile characterized by the absence of an immediate release phase, such as that obtained by IV administration.

As used herein, the term "T<max>" means the time point of maximum observed plasma concentration.

As used herein, the term "C<max>" means the maximum observed plasma concentration.

As used herein, the term "plasma t" means the observed "plasma half-life" and represents the time required for the plasma concentration of the drug to reach 50% of its maximum value (C<max>). This facilitates the determination of the mean duration of pharmacological effects. In addition, it facilitates direct and meaningful comparisons of the duration of different test articles after delivery by the same or different routes.

The term "optimal therapeutic targeting ratio" or "OTTR" represents the average time that the drug is present at therapeutic levels, defined as the time during which the plasma concentration of the drug remains above 50% of the C<max>normalized by the elimination half-life of the drug multiplied by the ratio between the C<max>obtained in the pharmaceutical form of interest and the C<max>after IV administration of equivalent doses and is calculated by the formula:

OTTR=(C<IVmax>/C<max>) x (Dose/Dose<IV>) (Time above 50% of C<max>)/(Terminal elimination half-life<IV>of the drug).

The term "substantially pure" means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard analytical methods, such as thin layer chromatography (TLC), gel electrophoresis, and high performance liquid chromatography (HPLC), used by those skilled in the art to assess such purity, or sufficiently pure that further purification will not detectably alter the physical or chemical properties of the compound. Methods for purifying compounds to yield substantially chemically pure compounds are known to those skilled in the art. However, a substantially chemically pure compound may be a mixture of stereoisomers or isomers. In such cases, further purification could increase the specific activity of the compound.

The term "substantially pure" when used with respect to enantiomers indicates that a particular enantiomer (e.g., an S-enantiomer or an R-enantiomer) is substantially free of its stereoisomer. In various embodiments, substantially pure indicates that a particular enantiomer is at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 98%, or at least 99% of the purified compound. Methods for producing substantially pure enantiomers are well known to those skilled in the art. For example, an individual stereoisomer, e.g., an enantiomer, substantially free of its stereoisomer may be obtained by resolution of the racemic mixture using a method such as the formation of diastereomers using optically active resolving agents (Stereochemistry of Carbon Compounds, (1962) by E. L. Eliel, McGraw Hill; Lochmuller (1975) J. Chromatogr., 113(3): 283-302). Racemic mixtures of chiral compounds may be separated and isolated by any suitable method including, but not limited to: (1) formation of ionic diastereomeric salts with chiral compounds and separation by fractional crystallization or other methods, (2) formation of diastereomeric compounds with chiral derivatization reagents, separation of the diastereomers and conversion to the pure stereoisomers, and (3) separation of the substantially pure or enriched stereoisomers directly under chiral conditions. Another approach to separation of enantiomers is to use a Diacel chiral column and elution using an organic mobile phase such as that performed by Chiral Technologies (www.chiraltech.com) on a fee-for-service basis.

Brief description of the drawings

Figure 1 illustrates various hydantoins.

Figure 2 illustrates various hydantoins.

Figure 3 illustrates proposed interaction models of the hydantoin with the FLAP region of BACE1. The bottom panel illustrates the interaction of the 3,4-substituent of ring B with FLAP, Trp76 disrupts the Trp-76-^Tyr-71H bond, causing Tyr-71 to flip to the left and interact with the difluoro-containing ring A.

Figure 4 illustrates the binding of the APP-binding BACE inhibitor (ABBI) FAH-3 to eAPP<575-624>as measured by surface plasmon resonance (SPR) screening. The binding affinity of compounds for the APP ectodomain was determined by SPR. A technique has been developed to measure the affinity of compounds for APP ectodomain fragments. For the binding experiments of compound 3 a TRX-eAPP575-624 substrate was used. eAPP was cross-linked to CM5 Biacore chips (GE Healthcare). Compound 3 was used at various concentrations in the flow through the chip and the plasmon resonance signal was determined using a Biacore T100.

Figure 5 illustrates the inhibition of Ap production by FAH-3.

Figure 6A illustrates the selectivity of ABBI for inhibiting APP-BACE cleavage compared to PSGL-BACE cleavage shown in Figure 6B.

Detailed description

In various embodiments, hydantoins are identified that appear to inhibit p-secretase-mediated processing of APP by a novel mechanism. In particular, without wishing to be bound by a particular theory, it is believed that these molecules interact with BACE and/or APP and/or a BACE/APP complex and thereby inhibit BACE cleavage of the APP substrate MBP-C125, resulting in inhibition of the production of C99 and the p-site peptide substrate (P5-P5'). Furthermore, the various hydantoins identified herein inhibit Ap42 in SHSY5Y neuroblastoma cells. Furthermore, it is shown that the activity of the hydantoins identified herein appears to be associated with binding to BACE and/or APP, particularly when these moieties form a BACE/APP complex. Therefore, the compounds described herein are believed to represent a new class of compounds designated herein as APP-binding BACE inhibitors (ABBIs) and provide a novel mechanism for modulating APP processing. The hydantoins described herein appear to exhibit enhanced brain permeability and functional inhibition of BACE.

ABBIs are specific for APP and/or BACE and/or the APP/BACE complex and are thought to exhibit fewer unwanted side effects because ABBIs are typically not active on other substrates for the enzyme or other enzyme complexes. With respect to Y-secretase inhibitors, non-APP substrates such as Notch raise concerns about potential side effects of Y-secretase inhibition and the recent failure of the Y-secretase inhibitor Semagacestat serves to reinforce such concerns. Similarly, in the case of BACE, for example, inhibition of non-APP substrates such as PSGL1 or LRP could lead to adverse side effects. Therefore, a desirable BACE inhibitor would be one that binds/interacts not with BACE but rather with APP, or with the APP/BACE complex leading to inhibition of the APP-specific BACE complex (ABBI).

Such ABBIs could potentially interact with the APP-BACE complex, for example, at the membrane, and prevent its transition to the "active" complex in early endosomes, where at pH < 5, BACE is fully active. Some p-site binding antibodies have been shown to block APP cleavage by BACE and also work in animal models of AD, however, for effective pharmaceutical development, small organic molecules are typically preferred over relatively large biomolecules such as antibodies.

The data reported here on the identification of the first ABBIs demonstrate that such an approach is feasible. Without wishing to be bound by a particular theory, ABBIs appear to inhibit BACE activity by interacting with APP, particularly when in an APP/BACE complex, thereby inhibiting BACE cleavage of amyloid precursor protein (APP), but not proteolytic cleavage of other substrates. Such therapies are thought to represent a new class of therapies for Alzheimer's disease (or other amyloidogenic diseases).

The active site of BACE1 is covered by flaps. A single flap 14 residues long forms a hairpin structure that is perpendicular to a cleft housing the active site and covers the central part of that active site. During the catalytic cycle, the flaps open to allow entry of the substrate (APP) into the catalytic cleft and also to release hydrolytic products. Initially, the hydantoins described herein were produced by introducing a dihalo (e.g., difluoro) ring into the aminohydantoin of Compound 0 (shown in Figure 1) to yield Compound 1 (also shown in Figure 1). Without wishing to be bound by a particular theory, it is thought that the dihalo ring introduces a FLAP interaction (e.g. an interaction with FLAP removes Tyr-71 (via pi stacking) and Trp-76 (via interaction with OCF2)) and restricts FLAP movement by limiting APP entry into the active site and/or egress of cleavage products (see e.g. Figure 3). This provides a novel family of small (MW <400) brain-penetrating BACE inhibitors (ABCIs).

Compound 2 and other hydantoins were produced (see, for example, compounds 1-5). Pharmacokinetic evaluation of these hydantoins was determined in brain uptake assays using NTg mice (see, for example, Table 1). Compound 1 was also found to reduce Ap42 in the same animals by 5 mpk, whereas Compound 3 reduced Ap by 1 mpk.

Table 1. Biological properties of illustrative hydantoins compared to BACE IV (Calbiochem p-secretase IV inhibitor cat. no. 565788.

*Calbiochem p-Secretase Inhibitor IV (Cat. #565788)

The compounds were also shown to interact with eAPP (see, for example, Figure 4) using a BiaCore assay. The hydantoins contemplated herein thus display desirable pharmacokinetic profiles and have the desired activity as demonstrated by interaction with APP and/or BACE/APP complexes and reduction of Ap42.

The sequential cleavage of APP by the membrane-bound proteases p-secretase and Y-secretase results in the formation of Ap. The p-site APP-cleaving enzyme 1 (BACE1) was identified as the primary p-secretase activity mediating the first cleavage of APP in the p-amyloidogenic pathway. In view of the ability of the ABBI compounds described herein to specifically block BACE1 activity on APP, it is believed (and the data presented herein show) that these ABBI compounds may reduce Ap levels or prevent the formation of neurotoxic Ap species. Accordingly, these compounds are thought to prevent or slow disease progression and/or prevent or slow the progression of preclinical manifestations of the amyloidogenic disease pathway.

Accordingly, it is believed that these agents (e.g., the hydantoins described herein, or one or more tautomers or stereoisomers thereof, or pharmaceutically acceptable salts or solvates of said one or more hydantoins, said one or more stereoisomers, or said one or more tautomers thereof) can be used to prevent or delay the onset of a pre-Alzheimer's cognitive dysfunction, and/or to ameliorate one or more symptoms of a pre-Alzheimer's cognitive dysfunction, and/or to prevent or delay the progression of a pre-Alzheimer's cognitive condition or dysfunction, and/or to promote the processing of amyloid precursor protein (APP) by the non-amyloidogenic pathway. In certain embodiments, these agents can be used in the treatment of Alzheimer's disease (e.g., to decrease the severity of the disease and/or ameliorate one or more symptoms of the disease and/or slow the progression of the disease).

Therapeutic and prophylactic methods.

In various embodiments, therapeutic and/or prophylactic methods are provided utilizing the one or more active agents (e.g., the hydantoins described herein, or one or more tautomers or stereoisomers thereof, or pharmaceutically acceptable salts or solvates of said one or more hydantoins, said one or more stereoisomers, or said one or more tautomers thereof). Typically, the methods involve administering one or more active agents to a subject (e.g., a human in need thereof) in an amount sufficient to achieve the desired therapeutic or prophylactic result.

Prophylaxis

In certain embodiments, the one or more active agents (e.g., the hydantoins described herein, or one or more tautomers or stereoisomers thereof, or pharmaceutically acceptable salts or solvates of said one or more hydantoins, said one or more stereoisomers, or said one or more tautomers thereof) are used in various prophylactic contexts. Thus, for example, in certain embodiments, the one or more active agents can be used to prevent or delay the onset of a pre-Alzheimer's cognitive dysfunction, and/or to ameliorate one or more symptoms of a pre-Alzheimer's cognitive condition and/or dysfunction, and/or to prevent or delay the progression of a pre-Alzheimer's cognitive condition and/or dysfunction to Alzheimer's disease.

Accordingly, in certain embodiments, the prophylactic methods described herein are contemplated for subjects identified as "at risk" and/or who have evidence of early pathological changes of Alzheimer's disease (AD), but who do not meet the clinical criteria for MCI or dementia. Without wishing to be bound by any particular theory, it is thought that even this “preclinical” stage of the disease represents a continuum of completely asymptomatic individuals with evidence of biomarkers suggestive of one or more AD pathophysiological processes (abbreviated as AD-P, see e.g. Sperling et al.(2011)Alzheimer’s & Dementia,1-13) at risk for progression to AD dementia to biomarker-positive individuals who are already demonstrating very subtle decline but who do not yet meet standardized criteria for MCI (see e.g. Albert et al.(2011)Alzheimer’s and Dementia,1-10 (doi:10.1016/j.jalz.2011.03.008).

This latter group of individuals could be classified as "non-normal, non-MCI" but could be referred to as "presymptomatic" or "preclinical" or "asymptomatic" or "premanifest." In various embodiments, this presymptomatic AD continuum may also encompass, but not necessarily not be limited to, (1) individuals carrying one or more apolipoprotein E (APOE) β4 alleles who are known or believed to be at increased risk for developing AD dementia, at the time they have positive biomarkers for AD-P, and (2) carriers of autosomal dominant mutations, who are in the biomarker-positive presymptomatic stage of their disease and who will almost certainly manifest clinical symptoms and progress to dementia.

A biomarker model has been proposed in which the most widely validated AD-P biomarkers become aberrant and also reach a threshold in an orderly manner (see e.g. Jack et al. (2010) Lancet Neurol.,9:119-128). This biomarker model parallels the proposed pathophysiological sequence of (pre-AD/AD), and is relevant for tracking preclinical (asymptomatic) stages of AD (see e.g. Figure 3 in Sperling et al. (2011) Alzheimer's & Dementia,1-13). Biomarkers of brain amyloidosis include, but are not limited to, reductions in Ap42 in CSF and increased amyloid tracer retention on positron emission tomography (PET) images. Elevated CSF tau is not specific to AD and is thought to be a biomarker of neuronal injury. Decreased 18F-fluorodeoxyglucose (FDG) uptake on PET with a temporoparietal pattern of hypometabolism is a biomarker of AD-related synaptic dysfunction. Brain atrophy on structural magnetic resonance imaging (MRI) in a characteristic pattern involving the medial temporal lobes, paralimbic and temporoparietal cortices is a biomarker of AD-related neurodegeneration. Other markers include, but are not limited to, volumetric MRI, FDG-PET, or plasma biomarkers (see, e.g., Vemurie et al.(2009)Neurology,73: 294-301; Yaffe et al.(2011)JAMA305: 261-266).

In certain embodiments, subjects suitable for the prophylactic methods contemplated herein include, but are not limited to, subjects characterized as having asymptomatic cerebral amyloidosis. In various embodiments, these individuals have evidence of biomarkers of Ap accumulation with elevated tracer retention on PET amyloid imaging and/or low Ap42 on CSF assay, but typically no detectable evidence of additional brain alterations suggestive of neurodegeneration or subtle cognitive and/or behavioral symptomatology.

It should be noted that currently available CSF and PET imaging biomarkers of Ap mainly provide evidence of amyloid accumulation and deposition of fibrillar forms of amyloid. The data suggest that soluble or oligomeric forms of Ap are likely in equilibrium with plaques, which may serve as reservoirs. In certain embodiments, it is contemplated that there is an identifiable preplaque stage where only soluble forms of Ap are present. In certain embodiments, it is contemplated that oligomeric forms of amyloid may be critical in the pathological cascade and provide useful markers. In addition, early synaptic changes may occur before there is evidence of amyloid accumulation.

In certain embodiments, subjects suitable for the prophylactic methods contemplated herein include, but are not limited to, subjects characterized as amyloid positive with evidence of synaptic dysfunction and/or early neurodegeneration. In various embodiments, these subjects have evidence of amyloid positivity and the presence of one or more markers of "downstream" AD-related neuronal injury. Illustrative, but not limiting, markers of neuronal injury include, but are not limited to, (1) elevated tau or phospho-tau in the CSF, (2) hypometabolism in an AD-like pattern (i.e., posterior cingulate, precuneus, and/or temporoparietal cortices) on FDG-PET, and (3) cortical thinning/gray matter loss in a specific anatomical distribution (i.e., lateral and medial parietal, posterior cingulate, and lateral temporal cortices) and/or hippocampal atrophy on volumetric MRI. Other markers include, but are not limited to, fMRI measures of default network connectivity. In certain embodiments, early synaptic dysfunction, as assessed by functional imaging techniques such as FDG-PET and fMRI, may be detectable prior to volumetric loss. Without wishing to be bound by a particular theory, it is believed that amyloid-positive individuals with evidence of early neurodegeneration may be further along the trajectory (i.e., in later stages of preclinical (asymptomatic) AD).

In certain embodiments, subjects suitable for the prophylactic methods contemplated herein include, but are not limited to, subjects characterized as amyloid-positive with evidence of neurodegeneration and subtle cognitive impairment. Without wishing to be bound by a particular theory, it is believed that those individuals with evidence of biomarkers of amyloid accumulation, early neurodegeneration, and evidence of subtle cognitive impairment are in the late stage of preclinical (asymptomatic) AD and are approaching the borderline with clinical criteria for mild cognitive impairment (MCI). These individuals may demonstrate evidence of decline from their own baseline (particularly when indicators of cognitive reserve are taken into consideration), even if they still perform within the "normal" range on standard cognitive measures. Without wishing to be bound by a particular theory, it is believed that more sensitive cognitive measures, particularly with challenging measures of episodic memory, may be able to detect very subtle cognitive impairment in amyloid-positive individuals. In certain embodiments, criteria include, but are not limited to, self-reported memory impairment or other subtle neurobehavioral changes.

As noted above, subjects/patients amenable to the prophylactic methods described herein include individuals at risk for disease (e.g., a pathology characterized by amyloid plaque formation such as MCI) but not exhibiting symptoms, as well as subjects currently exhibiting certain symptoms or markers. The risk for MCI and subsequent Alzheimer's disease is known to generally increase with age. Accordingly, in asymptomatic subjects with no other known risk factors, in certain embodiments, prophylactic application is contemplated for subjects over the age of 50, or subjects over the age of 55, or subjects over the age of 60, or subjects over the age of 65, or subjects over the age of 70, or subjects over the age of 75, or subjects over the age of 80, in particular to prevent or delay the onset or ultimate severity of mild cognitive impairment (MCI), and/or to slow or prevent the progression of MCI to early stage Alzheimer's disease (AD).

In certain embodiments, the methods described herein are especially useful for individuals who have a known genetic risk for Alzheimer's disease (or other amyloidogenic pathologies), whether they are asymptomatic or exhibit symptoms of the disease. Such individuals include those who have relatives who have experienced MCI or AD (e.g., a parent, grandparent, sibling) and those whose risk is determined by analysis of genetic or biochemical markers. Genetic markers of risk for Alzheimer's disease include, for example, mutations in the APP gene, particularly mutations at position 717 and at positions 670 and 671 referred to as the Hardy and Swedish mutations, respectively (see Hardy (1997) Trends. Neurosci, 20: 154-159). Other risk markers include mutations in the presenilin genes (PS1 and PS2), family history of AD, having the familial Alzheimer's disease (FAD) mutation, the APOE £4 allele, hypercholesterolemia, or atherosclerosis. Additional susceptibility genes for determining the development of Alzheimer's disease are reviewed, for example, in Sleegers,et al.(2010)Trends Genet.26(2):84-93.

In some embodiments, the subject is asymptomatic but has familial and/or genetic risk factors for developing MCI or Alzheimer's disease. In asymptomatic patients, treatment may begin at any age (e.g., about age 20, about age 30, about age 40, about age 50). Typically, however, treatment need not begin until the patient reaches at least about age 40, or at least about age 50, or at least about age 55, or at least about age 60, or at least about age 65, or at least about age 70.

In some embodiments, the subject exhibits symptoms of, for example, mild cognitive impairment (MCI) or Alzheimer's disease (AD). Individuals currently suffering from Alzheimer's disease can be recognized by the characteristic dementia, as well as the presence of the risk factors described above. In addition, several diagnostic tests are available to identify individuals who have AD. These include measurement of CSF levels of Tau, phospho-tau (pTau), Ap42, and C-terminally cleaved APP fragment (APPneo). Increased total Tau (tTau), phospho-Tau (pTau), APPneo, soluble Ap40, pTau/Ap42 ratio, and tTau/Ap42 ratio, and decreased Ap42 levels, Ap42/Ap40 ratio, Ap42/Ap38 ratio, sAPPa levels, sAPPa/sAPPp ratio, sAPPa/Ap40 ratio, and sAPPa/Ap42 ratio signify the presence of AD. In some embodiments, the subject or patient is diagnosed with MCI. Elevated levels of neural chain protein (NTP) in urine and/or elevated levels of a2-macroglobulin (a2M) and/or complement factor H (CFH) in plasma are also biomarkers of MCI and/or AD (see, for example, Anoopet al.(2010)Int. J. Alzheimer's Dis.2010:606802).

In certain embodiments, subjects amenable to treatment may have age-related memory impairment (AMD) or mild cognitive impairment (MCI). The methods described herein are particularly suitable for the prophylaxis and/or treatment of MCI. In such cases, the methods may delay or prevent the onset of MCI, and/or reduce one or more characteristic symptoms of MCI and/or delay or prevent the progression of MCI to early, middle, or late stage Alzheimer's disease or reduce the ultimate severity of the disease.

Mild cognitive impairment (MCI)

Mild cognitive impairment (MCI, also known as incipient dementia or isolated memory impairment) is a diagnosis given to individuals who have cognitive impairments beyond that expected for their age and education but which typically do not significantly interfere with daily activities (see, for example, Petersen et al.(1999)Arch. Neurol.56(3):303-308). In many cases it is considered a borderline or transitional stage between normal aging and dementia. Although MCI can present with a variety of symptoms, when memory loss is the predominant symptom it is called "amnestic MCI" and is often considered a risk factor for Alzheimer's disease (see, for example, Grundman et al.(2004)Arch. Neurol.61(1):59-66; and on the Internet at en.wikipedia.org/wiki/Mild_cognitive_impairment - cite_note-Grundman-1). When individuals have impairments in domains other than memory, they are often classified as single- or multiple-domain non-amnestic MCI and these individuals are thought to be more likely to develop other dementias (e.g., dementia with Lewy bodies). There is evidence to suggest that while patients with amnestic MCI may not meet neuropathological criteria for Alzheimer's disease, patients may be in a transitional stage in the evolution of Alzheimer's disease; patients in this hypothesized transitional stage demonstrated diffuse amyloid in the neocortex and frequent neurofibrillary tangles in the medial temporal lobe (see, e.g., Petersen et al. (2006) Arch. Neurol. 63(5): 665-72).

The diagnosis of MCI typically involves a comprehensive clinical evaluation including clinical observation, neuroimaging, blood tests, and neuropsychological testing. In certain embodiments, the diagnostic criteria for MCI include, but are not limited to, those described by Albert et al. (2011) Alzheimer's & Dementia.1-10 As described therein, the diagnostic criteria include (1) basic clinical criteria that could be used by healthcare providers without access to advanced imaging techniques or cerebrospinal fluid analysis, and (2) research criteria that could be used in clinical research settings, including clinical trials. The second set of criteria incorporates the use of imaging-based biomarkers and cerebrospinal fluid measurements. The final set of criteria for mild cognitive impairment due to AD has four levels of certainty, depending on the presence and nature of the biomarker findings.

In certain embodiments, the clinical evaluation/diagnosis of MCI involves: (1) Concern for reflecting a change in cognition reported by the patient, informant, or physician (i.e., historical or observed evidence of decline over time); (2) Objective evidence of decline in one or more cognitive domains, typically including memory (i.e., formal or bedside testing to establish level of cognitive function across multiple domains); (3) Preservation of independence in functional abilities; (4) Not demented; and in certain embodiments, (5) An etiology of MCI consistent with pathophysiological processes of AD. Typically vascular, traumatic, and medical causes of cognitive decline are ruled out where possible. In certain embodiments, where possible, evidence of longitudinal decline in cognition is identified. The diagnosis is further strengthened by a history consistent with genetic factors for AD, where relevant.

With respect to decline in one or more cognitive domains, there should be evidence of concern for a change in cognition, compared to the person's previous level. There should be evidence of poorer performance in one or more cognitive domains that is greater than would be expected for the patient's age and educational level. If repeated assessments are available, then a decline in performance should be apparent over time. This change can take place across a variety of cognitive domains, including memory, executive function, attention, language, and visuospatial skills. Impairment of episodic memory (i.e., the ability to learn and retain new information) is most often seen in patients with MCI who subsequently progress to a diagnosis of AD dementia.

With respect to maintaining independence in functional abilities, it is noted that individuals with MCI commonly have mild problems performing complex functional tasks that they used to use for shopping. They may take longer, be less efficient, and make more errors when performing such activities than in the past. However, they generally maintain their functional independence in daily life, with minimal help or assistance.

With respect to dementia, cognitive changes must be mild enough that there is no evidence of significant decline in social or occupational functioning. If an individual has been assessed only once, the change will be inferred from history and/or evidence that cognitive performance is impaired beyond what would have been expected for that individual.

Cognitive testing is optimal for objectively assessing an individual's degree of cognitive impairment. Scores on cognitive tests for individuals with MCI typically fall between 1 and 1.5 standard deviations below the mean of their age- and education-matched peers on culturally appropriate normative data (i.e., for the one or more impaired domains, when available).

Episodic memory (i.e., the ability to learn and retain new information) is most often seen in patients with MCI who subsequently progress to a diagnosis of AD dementia. There are a variety of episodic memory tests that are useful in identifying those patients with MCI who have a high likelihood of progressing to AD dementia within a few years. These tests typically assess immediate and delayed recall, so that it is possible to determine retention over a delay. Many, but not all, of the tests that have been shown to be useful in this regard are word list learning tests with multiple trials. These tests reveal the rate of learning over time as well as the maximum amount acquired over the course of the learning trials. They are also useful in demonstrating that the individual is, in fact, attending to the task in immediate recall, which can then be used as a benchmark for assessing the relative amount of material retained in delayed recall. Examples of such tests include (but are not limited to): the Free and Cued Selective Recall Test, the Rey Auditory Verbal Learning Test, and the California Verbal Learning Test. Other measures of episodic memory include, but are not limited to: immediate and delayed recall of a paragraph such as the Logical Memory I and II of the Wechsler Memory Scale-Revised (or other versions) and immediate and delayed recall of nonverbal materials such as the Visual Reproduction subtests of the Wechsler Memory Scale-Revised I and II.

Because other cognitive domains may be impaired among individuals with MCI, it is desirable to examine domains in addition to memory. These include, but are not limited to, executive functions (e.g., task switching, reasoning, problem solving, planning), language (e.g., naming, fluency, expressive speech, and comprehension), visuospatial skills, and attention control (e.g., single and divided attention). There are many clinical neuropsychological measures available to assess these cognitive domains, including (but not limited to) the Trace Test (executive function), the Boston Naming Test, letter-category fluency (language),

As noted above, genetic factors may be incorporated into the diagnosis of MCI. If an autosomal dominant form of AD is known to be present (i.e., mutation in APP, PS1, PS2), then the development of MCI is most likely the precursor to AD dementia. The vast majority of these cases develop early-onset AD (i.e., onset before age 65).

Furthermore, there are genetic influences on the development of late-onset AD dementia. For example, the presence of one or two £4 alleles in the apolipoprotein E (APOE) gene is a widely accepted genetic variant as conferring increased risk for late-onset AD dementia. Evidence suggests that an individual who meets the clinical, cognitive, and etiologic criteria for MCI, and is also APOE £4 positive, is more likely to progress to AD dementia within a few years than an individual without this genetic feature. Additional genes are thought to play an important, but minor, role than APOE and also confer changes in the risk of progression to AD dementia (see, for example, Bertramet et al.(2010)Neuron,21:270-281).

In certain embodiments, subjects suitable for the prophylactic methods described herein include, but are not limited to, subjects identified with one or more of the basic clinical criteria described above and/or subjects identified with one or more "investigational criteria" for MCI, for example, as described below.

"Investigational criteria" for the identification/prognosis of MCI include, but are not limited to, biomarkers that increase the likelihood that the MCI syndrome is due to the pathophysiological processes of AD. Without wishing to be bound by any particular theory, it is believed that the joint application of clinical criteria and biomarkers may result in varying levels of certainty that the MCI syndrome is due to the pathophysiological processes of AD. In certain embodiments, two categories of biomarkers are contemplated that have been most widely studied and applied to clinical outcomes. These include "Ap" (including Ap42 in CSF and/or amyloid PET imaging) and "biomarkers of neuronal injury" (including, but not limited to, tau/p-tau in CSF, hippocampal or medial temporal lobe atrophy on MRI, and temporoparietal/precuneus hypometabolism or hypoperfusion on PET or SPECT).

Without wishing to be bound by a particular theory, it is thought that evidence of both Ap and neuronal injury (either an increase in tau/p-tau or imaging biomarkers in a characteristic AD topographic pattern) together confer the highest likelihood that the AD pathophysiological process is present. Conversely, if these biomarkers are negative, this may provide information about the likelihood of an alternative diagnosis. It is recognized that biomarker findings may be contradictory and therefore any combination of biomarkers is indicative (an indicator) used in the context of a differential diagnosis and not determinative in itself. It is recognized that different degrees of severity of an abnormality may confer different probabilities or prognoses, which are difficult to accurately quantify for broad application.

For those potential MCI subjects whose clinical and cognitive syndrome of MCI is consistent with AD as the etiology, the addition of biomarker analysis affects the levels of certainty in the diagnosis. In the most typical example where the clinical and cognitive syndrome of MCI has been established, including evidence of an episodic memory disorder and a presumed degenerative etiology, the most likely cause is the neurodegenerative process of AD. However, the final outcome still has varying degrees of certainty. The likelihood of progression to AD dementia will vary depending on the severity of cognitive impairment and the nature of the evidence suggesting that AD pathophysiology is the underlying cause. Without wishing to be bound by a particular theory, it is thought that positive biomarkers reflecting neuronal injury increase the likelihood of progression to dementia occurring within a few years, and that positive findings reflecting both Ap accumulation and neuronal injury together confer the highest likelihood that the diagnosis is MCI due to AD.

A positive Ap biomarker and a positive neuronal injury biomarker provide an indication that the MCI syndrome is due to AD processes and that the subject is well suited for the methods described herein.

A positive Ap biomarker in a situation where biomarkers of neuronal injury have not been or cannot be assayed or a positive biomarker of neuronal injury in a situation where Ap biomarkers have not been or cannot be assayed indicates an intermediate likelihood that the MCI syndrome is due to AD. Such subjects are believed to be well suited for the methods described herein.

Negative biomarkers for both Ap and neuronal injury suggest that the MCI syndrome is not due to AD. In such cases, subjects may not be suitable for the methods described herein.

There is evidence that MRI can observe deterioration, including progressive loss of grey matter in the brain, from mild cognitive impairment to full-blown Alzheimer's disease (see, for example, Whitwell et al.(2008)Neurology70(7):512-520). A technique known as PiB PET imaging is used to clearly show the sites and shapes of amyloid beta deposits in living subjects using a C11 tracer that selectively binds to such deposits (see, for example, Jacket et al.(2008)Brain13 1(Pt 3):665-680).

In certain embodiments, MCI is typically diagnosed when there is 1) evidence of memory impairment; 2) preservation of general cognitive and functional abilities; and 3) absence of diagnosed dementia.

In certain embodiments, MCI and Alzheimer's disease stages may be identified/categorized, in part, by Clinical Dementia Rating (CDR) scores. The CDR is a five-point scale used to characterize six domains of cognitive and functional performance applicable to Alzheimer's disease and related dementias: Memory, Orientation, Judgment and Problem Solving, Activities Outside the Home, Household Activities and Hobbies, and Self-Care. Information to make each rating may be obtained through a semi-structured interview of the patient and a reliable informant or collateral source (e.g., a family member).

The CDR table provides descriptive anchors that guide the clinician in making appropriate ratings based on interview data and clinical judgment. In addition to ratings for each domain, an overall CDR score can be calculated using an algorithm. This score is useful for characterizing and tracking a patient's level of impairment/dementia: 0 = Normal; 0.5 = Very mild dementia; 1 = Mild dementia; 2 = Moderate dementia; and 3 = Severe dementia. An illustrative CDR table is shown in Table 2.

Table 2. Illustrative table of the CDR clinical classification of dementia.

continued

A CDR score of ~0.5 or ~0.5 to 1.0 is often considered clinically relevant MCI. Higher CDR scores may be indicative of progression to Alzheimer's disease.

In certain embodiments, administration of one or more agents described herein (e.g., the hydantoins described herein, or one or more tautomers or stereoisomers thereof, or pharmaceutically acceptable salts or solvates of said one or more hydantoins, said one or more stereoisomers, or said one or more tautomers thereof) is considered effective when there is a reduction in the CSF of the levels of one or more components selected from the group consisting of Tau, phospho-Tau (pTau), APPneo, soluble Ap40, soluble Ap42, and/or the ratio of Ap42/Ap40, and/or when there is a reduction in the plaque burden in the brain of the subject, and/or when there is a reduction in the rate of plaque formation in the brain of the subject, and/or when there is an improvement in the cognitive abilities of the subject, and/or when there is a perceived improvement in the quality of life by the subject, and/or when there is a reduction in the incidence of stroke, stroke, or stroke in the brain of the subject. significant in clinical dementia rating (CDR), and/or when the rate of increase in clinical dementia rating slows or stops and/or progression from MCI to early-stage AD when it slows or stops.

In some embodiments, a diagnosis of MCI can be determined by considering the results of several clinical tests. For example, Grundman, et al. (2004) Arch Neurol 61: 59-66, report that a diagnosis of MCI can be clinically efficiently established using a simple memory test (paragraph recall) to establish an objective memory deficit, a measure of general cognition (Mini-Mental State Examination (MMSE), discussed in greater detail below) to exclude broader cognitive impairment beyond memory, and a structured clinical interview (CDR) with patients and caregivers to verify the patient's memory complaint and memory loss and to ensure that the patient is not demented. Patients with MCI perform, on average, less than 1 standard deviation (S.D.) below normal on the non-memory cognitive measures included in the battery. Tests of learning, attention, perceptual speed, category fluency, and executive function may be impaired in patients with MCI, but are much less prominent than memory deficits.

Alzheimer's disease (AD).

In certain embodiments, the one or more active agents (e.g., the hydantoins described herein, or one or more tautomers or stereoisomers thereof, or pharmaceutically acceptable salts or solvates of said one or more hydantoins, said one or more stereoisomers, or said one or more tautomers thereof) are contemplated for the treatment of Alzheimer's disease. In such cases, the methods described herein are useful for preventing or slowing the onset of Alzheimer's disease (AD), for reducing the severity of AD when the subject has transitioned to the clinical diagnosis of AD, and/or for mitigating one or more symptoms of Alzheimer's disease.

In particular, when Alzheimer's disease is in an early stage, the methods may reduce or eliminate one or more characteristic symptoms of AD and/or delay or prevent the progression of MCI to early- or late-stage Alzheimer's disease.

Individuals who currently have Alzheimer's disease can be recognized by the characteristic dementia as well as the presence of the risk factors described above. In addition, several diagnostic tests are available to identify people who have AD. Individuals who currently have Alzheimer's disease can be recognized by the characteristic dementia as well as the presence of the risk factors described above. In addition, several diagnostic tests are available to identify people who have AD. These include measurement of CSF levels of Tau, phospho-tau (pTau), sAPPa, sAPPp, Ap40, Ap42, and/or the C-terminally cleaved APP fragment (APPneo). Increased Tau, pTau, sAPPp, and/or APPneo, and/or decreased levels of sAPPa, soluble Ap40, and/or soluble Ap42, particularly in the context of a differential diagnosis, may signify the presence of AD.

In certain embodiments, subjects amenable to treatment may have Alzheimer's disease. Individuals suffering from Alzheimer's disease may also be diagnosed according to the Alzheimer's Disease and Related Disorders Association (ADRDA) criteria. The NINCDS-ADRDA Alzheimer's criteria were proposed in 1984 by the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association (now known as the Alzheimer's Association) and are among the most widely used in the diagnosis of Alzheimer's disease (AD). McKhann,et al.(1984)Neurology34(7):939-44. According to these criteria, the presence of cognitive impairment and a suspected dementia syndrome must be confirmed by neuropsychological testing for a clinical diagnosis of possible or probable AD. However, histopathological confirmation (microscopic examination of brain tissue) is generally used for a decisive diagnosis. The NINCDS-ADRDA Alzheimer's criteria specify eight cognitive domains that may be affected in AD: memory, language, perceptual skills, attention, constructive skills, orientation, problem solving, and functional skills. These criteria have shown good reliability and validity.

Baseline assessments of patient function can be made using classical psychometric measures such as the Mini-Mental State Examination (MMSE) (Folstein et al. (1975) J. Psychiatric Research 12 (3): 189-198), and the Alzheimer's Disease Rating Scale (ADAS), which is a comprehensive scale for assessing the status and function of patients with Alzheimer's disease (see, for example, Rosen, et al. (1984) Am. J. Psychiatr., 141: 1356-1364). These psychometric scales provide a measure of the progression of Alzheimer's disease. Qualitative life scales suitable for monitoring treatment can also be used. The degree of disease progression can be determined using a Mini-Mental State Examination (MMSE) (see, for example, Folstein, et al. cited above). Any score greater than or equal to 25 points (out of 30) is effectively normal (intact). Below this, scores may indicate severe (<9 points), moderate (10-20 points) or mild (21-24 points) Alzheimer's disease.

Alzheimer's disease can be divided into several stages, including: 1) Moderate cognitive impairment (mild or early-stage Alzheimer's disease), 2) Moderately severe cognitive impairment (moderate or mid-stage Alzheimer's disease), 3) Severe cognitive impairment (moderately severe or mid-stage Alzheimer's disease), and 4) Very severe cognitive impairment (severe or late-stage Alzheimer's disease) as shown in Table 3.

Table 3. Illustrative stages of Alzheimer's disease.

continued

In various embodiments, administration of one or more agents described herein to subjects diagnosed with Alzheimer's disease is considered effective when there is a reduction in the CSF levels of one or more components selected from the group consisting of Tau, phospho-Tau (pTau), APPneo, soluble Ap40, soluble Ap42, and/or the ratio of Ap42/Ap40, and/or when there is a reduction in the plaque load in the subject's brain, and/or when there is a reduction in the rate of plaque formation in the subject's brain, and/or when there is an improvement in the subject's cognitive abilities, and/or when there is a perceived improvement in the subject's quality of life, and/or when there is a significant reduction in the subject's clinical dementia rating (CDR), and/or when the rate of increase in the clinical dementia rating slows or stops and/or the progression of AD slows or stops (e.g., when the transition from one stage to another as listed below). in Table 3 slows down or stops).

In certain embodiments, subjects amenable to the present methods generally do not have a neurological disease or disorder other than Alzheimer's disease. For example, in certain embodiments, the subject does not have or is not at risk of developing a neurological disease or disorder, such as Parkinson's disease and/or schizophrenia and/or psychosis.

Active agent or agents.

The methods described herein are based, in part, on the discovery that administration of one or more active agents (e.g., the hydantoins described herein, or one or more tautomers or stereoisomers thereof, or pharmaceutically acceptable salts or solvates of said one or more hydantoins, said one or more stereoisomers, or said one or more tautomers thereof) find use in the treatment and/or prophylaxis of diseases characterized by amyloid deposits in the brain, e.g., mild cognitive impairment, Alzheimer's disease, macular degeneration, and the like.

In certain embodiments, the compound is a compound according to Formula XVII:

X V II (FAH-6).

In certain embodiments, the compound is a compound according to Formula XVIII:

In certain embodiments, the compound is a compound according to Formula XIX:

In certain embodiments, the compound is a compound according to Formula XXI:

In certain embodiments, the compound is a compound according to Formula XXIII:

In certain embodiments, the compound is a compound according to Formula XXVI:

In certain embodiments, the compound is a compound according to Formula XXVII:

In certain embodiments, the compound is a compound according to Formula XXVIII:

In certain embodiments, the compound is a compound according to Formula XXIX:

In certain embodiments, the compound is a compound according to Formula XXXI:

In certain embodiments, the compound is a compound according to Formula XXXII:

or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of a tautomer thereof, an enantiomer thereof, or a pharmaceutically acceptable salt of an enantiomer thereof.

In certain embodiments, any of the above compounds is a substantially pure S-enantiomer. In certain embodiments, any of the above compounds is a substantially pure R-enantiomer. Various compounds contemplated herein include the compounds shown in Table 4, namely, FAH-2, FAH-4, FAH-5, FAH-6, FAH-9, FAH-10, FAH-12, FAH-14, FAH-19, FAH-22, FAH-23, FAH-25, FAH-28, with the others being presented for illustrative purposes only.

Table 4. Illustrative compounds

(continued)

(continued)

(continued)

(continued)

With respect to these compounds, pharmaceutically acceptable salts, tautomers, pharmaceutically acceptable salts of a tautomer, enantiomers thereof, and pharmaceutically acceptable salts of an enantiomer thereof are also contemplated. In addition, substantially pure S-enantiomers or substantially pure R-enantiomers of these compounds are contemplated.

Various illustrative hydantoins are also shown in Figures 1 and 2. In certain embodiments, pharmaceutically acceptable salts, tautomers, pharmaceutically acceptable salts of a tautomer, enantiomers thereof, and a pharmaceutically acceptable salt of an enantiomer are contemplated.

With respect to certain molecules in Figure 1, without wishing to be bound by any particular theory, it is believed that the methylated OCHF<2>B ring shows increased potency with a 3,4-substitution. This type of substitution pattern is thought to interact with Trp-76 of the BACE flap, disrupting the interaction of Tyr-71 of the flap with Trp-76 and flipping Tyr-71 to the left, allowing it to interact with the difluoro groups of the A ring (see also Figure 3).

In certain embodiments, the compound is a substantially pure "S" enantiomer. In certain embodiments, the compound is a substantially pure "R" enantiomer. In certain embodiments, the compound binds to APP and/or the BACE enzyme and/or an APP/BACE complex.

Methods for preparing hydantoins such as those described herein are known to those skilled in the art. Generally, in one approach, the relevant hydantoin (e.g., a difluorohydantoin) would be prepared from 3,4-difluorobenzaldehyde converted to dione and condensed with urea to produce the hydantoin as described in Example 1.

Illustrative protocols for the synthesis of FAH-1, FAH-2, FAH-3, FAH-4, FAH-5, FAH-17, FAH-17 HCl salt, FAH-22, FAH-23, FAH-27, and FAH-28 (see Table 4) are provided in Examples 1-11. The syntheses of additional compounds described herein are simple variations of the synthetic schemes provided herein.

The various active agents and synthetic schemes are intended to be illustrative and not limiting. Using the teachings provided herein, many others (e.g., hydantoins or one or more tautomers or stereoisomers thereof, or pharmaceutically acceptable salts or solvates of said one or more hydantoins, said one or more stereoisomers, or said one or more tautomers thereof, can be synthesized and identified by one of skill in the art.

Illustrative activity of selected APP-selective BACE inhibitors described above is shown in Table 5.

Table 5. Illustrative activity of certain APP-selective BACE inhibitors described herein.

Pharmaceutical formulations.

In certain embodiments, one or more active agents described herein (e.g., the hydantoins described herein, or one or more tautomers or stereoisomers thereof, or pharmaceutically acceptable salts or solvates of said one or more hydantoins, said one or more stereoisomers, or said one or more tautomers thereof) are administered to a mammal in need thereof, for example, a mammal at risk for or suffering from a pathology characterized by abnormal processing of amyloid precursor proteins, a mammal at risk for progression from MCI to Alzheimer's disease, etc. In certain embodiments, the one or more active agents are administered to prevent or delay the onset of a pre-Alzheimer's cognitive condition and/or dysfunction, and/or to improve one or more symptoms of a pre-Alzheimer's cognitive dysfunction, and/or to prevent or delay the progression of a pre-Alzheimer's cognitive condition or dysfunction to Alzheimer's disease, and/or to promote the processing of amyloid precursor protein (APP) by a non-amyloidogenic pathway.

In certain embodiments, one or more active agents described herein (e.g., the hydantoins described herein, or one or more tautomers or stereoisomers thereof, or pharmaceutically acceptable salts or solvates of said one or more hydantoins, said one or more stereoisomers, or said one or more tautomers thereof) are administered to a mammal in need thereof, for example, a mammal at risk for or suffering from a pathology characterized by abnormal processing of amyloid precursor proteins in conditions other than Alzheimer's disease or MCI. Illustrative conditions include, but are not limited to, AD-type symptoms of patients with Down syndrome, glaucoma, macular degeneration (e.g., age-related macular degeneration (AMD), olfactory impairment, in the treatment of type II diabetes including diabetes associated with amyloidogenesis, neurodegenerative diseases such as sheep scrapie, bovine spongiform encephalopathies (e.g., BSE), traumatic brain injury ("TBI"), Creutzfeld-Jakob disease, and the like, type II diabetes. Other conditions characterized by amyloid formation/deposition are contemplated. Such conditions include, but are not limited to, Huntington's disease, medullary carcinoma of the thyroid, cardiac arrhythmias, isolated atrial amyloidosis, atherosclerosis, rheumatoid arthritis, aortic medial amyloid, prolactinomas, familial amyloid polyneuropathy, hereditary non-neuropathic systemic amyloidosis, dialysis-related amyloidosis, Finnish-type amyloidosis, lattice corneal dystrophy, cerebral amyloid angiopathy (e.g., Icelandic type), systemic AL amyloidosis, sporadic inclusion body myositis, cerebrovascular dementia, and the like.

The one or more active agents (e.g., the hydantoins described herein) may be administered in "native" form or, if desired, in the form of salts, esters, amides, derivatives and the like, provided that the salt, ester, amide or derivative is pharmacologically suitable, i.e., effective in the present methods. Salts, esters, amides and other derivatives of the active agents may be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry and described, for example, by March (1992) Advanced Organic Chemistry; Reactions, Mechanisms and Structure, 4th Ed. N.Y. Wiley-Interscience, and as described above.

For example, a pharmaceutically acceptable salt may be prepared for any of the one or more agents described herein having functionality capable of forming a salt. A pharmaceutically acceptable salt is any salt that retains the activity of the parent compound and does not impart any harmful or adverse effect on the subject to which it is administered and in the context in which it is administered.

In various embodiments, pharmaceutically acceptable salts may be obtained from organic or inorganic bases. The salt may be a mono- or polyvalent ion. Of particular interest are the inorganic ions lithium, sodium, potassium, calcium and magnesium. Organic salts may be prepared with amines, particularly ammonium salts such as mono-, di- and trialkylamines or ethanolamines. Salts may also be formed with caffeine, tromethamine and similar molecules.

Methods for formulating pharmaceutically active agents such as salts, esters, amides and the like are well known to those skilled in the art. For example, salts can be prepared from the free base using conventional methodology which typically involves reaction with a suitable acid. Generally, the basic form of the drug is dissolved in a polar organic solvent such as methanol or ethanol and the acid is added. The resulting salt either precipitates or can be brought out of solution by addition of a less polar solvent. Suitable acids for preparing acid addition salts include, but are not limited to, both organic acids, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like, as well as inorganic acids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like. An acid addition salt can be reconverted to the free base by treatment with a suitable base. Certain particularly preferred acid addition salts of the active ingredients of the present invention include halide salts, such as those that can be prepared using hydrochloric or hydrobromic acids. On the contrary, the preparation of basic salts of the active ingredients of this invention is prepared in a similar manner using a pharmaceutically acceptable base such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or the like. Particularly preferred basic salts include alkali metal salts, for example, the sodium salt and copper salts.

For the preparation of salt forms of basic drugs, the pKa of the counterion is preferably at least about 2 pH units lower than the pKa of the drug. Similarly, for the preparation of salt forms of acidic drugs, the pKa of the counterion is preferably at least about 2 pH units higher than the pKa of the drug. This allows the counterion to drive the pH of the solution below pHmax to reach the salt plateau, where the solubility of the salt prevails over the solubility of the free acid or base. The generalized rule of difference in pKa units of the ionizable group in the active pharmaceutical ingredient (API) and in the acid or base is intended to make proton transfer energetically favorable. When the pKa of the API and counterion are not significantly different, a solid complex may form, but it may rapidly disproportionate (i.e., break down into the individual drug and counterion entities) in an aqueous environment.

Preferably, the counterion is a pharmaceutically acceptable counterion. Suitable anionic salt forms include, but are not limited to, acetate, benzoate, benzylate, bitartrate, bromide, carbonate, chloride, citrate, edetate, edisylate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, napsylate, nitrate, pamoate (embonate), phosphate and diphosphate, salicylate and disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethiodide, valerate, and the like, while suitable cationic salt forms include, but are not limited to, aluminum, benzathine, calcium, ethylenediamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine, zinc, and the like.

The preparation of esters typically involves the functionalization of hydroxyl and/or carboxyl groups that are present within the molecular structure of the active ingredient. In certain embodiments, esters are typically acyl substituted derivatives of free alcohol groups, i.e., moieties derived from carboxylic acids of the formula RCOOH, where R is alkyl and is preferably lower alkyl. Esters may be reconverted to free acids, if desired, by using conventional hydrogenolysis or hydrolysis procedures.

Amides may also be prepared using techniques known to those skilled in the art or described in the relevant literature. For example, amides may be prepared from esters, using suitable amine reagents, or may be prepared from an anhydride or an acid chloride by reaction with ammonia or a lower alkyl amine.

In various embodiments, the active agents identified herein (e.g., the hydantoins described herein, or one or more tautomers or stereoisomers thereof, or pharmaceutically acceptable salts or solvates of said one or more hydantoins, said one or more stereoisomers, or said one or more tautomers thereof) are useful for parenteral administration, topical administration, oral administration, nasal (or otherwise inhaled) administration, rectal administration, or local administration, such as by aerosol or transdermal, for the prophylactic and/or therapeutic treatment of one or more of the pathologies/indications described herein (e.g., pathologies characterized by excess amyloid plaque formation and/or deposition or unwanted amyloid or pre-amyloid processing).

In various embodiments, the active agents described herein may also be combined with a pharmaceutically acceptable carrier (excipient) to form a pharmacological composition. Pharmaceutically acceptable carriers may contain one or more physiologically acceptable compounds that act, for example, to stabilize the composition or to increase or decrease the absorption of the one or more active agents. Physiologically acceptable compounds may include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, protection and absorption enhancers such as lipids, compositions that reduce the clearance or hydrolysis of the active agents, or excipients or other stabilizers and/or buffers.

Other physiologically acceptable compounds, particularly for use in the preparation of tablets, capsules, gel capsules and the like include, but are not limited to, binders, diluents/fillers, disintegrants, lubricants, suspending agents and the like.

In certain embodiments, to manufacture an oral dosage form (e.g., a tablet), an excipient (e.g., lactose, sucrose, starch, mannitol, etc.), an optional disintegrant (e.g., calcium carbonate, calcium carboxymethylcellulose, sodium starch glycolate, crospovidone, etc.), a binder (e.g., alpha-starch, acacia, microcrystalline cellulose, carboxymethylcellulose, polyvinylpyrrolidone, hydroxypropylcellulose, cyclodextrin, etc.), and an optional lubricant (e.g., talc, magnesium stearate, polyethylene glycol 6000, etc.), for example, are added to the active component(s) (e.g., the hydantoins described herein, or one or more tautomers or stereoisomers thereof, or pharmaceutically acceptable salts or solvates of said one or more hydantoins, said one or more stereoisomers, or said one or more tautomers or stereoisomers thereof). or more tautomers thereof) and the resulting composition is compressed. Where necessary, the compressed product is coated, for example, using known methods for taste masking or for enteric dissolution or sustained release. Suitable coating materials include, but are not limited to, ethylcellulose, hydroxymethylcellulose, POLYOX® ethylene glycol, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate and Eudragit (Rohm & Haas, Germany; methacrylic-acrylic copolymer).

Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives which are particularly useful in preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. One skilled in the art would appreciate that the choice of one or more pharmaceutically acceptable carriers, including a physiologically acceptable compound, depends, for example, on the route of administration of the one or more active agents and the particular physicochemical characteristics of the one or more active agents.

In certain embodiments, the excipients are sterile and generally free of unwanted matter. These compositions may be sterilized by conventional sterilization techniques known in the art. For various oral dosage forms, excipients such as tablets and capsules, sterility is not required. Typically, the application of the USP/NF standard is sufficient.

Pharmaceutical compositions may be administered in a variety of unit dosage forms depending on the method of administration. Suitable unit dosage forms include, but are not limited to, powders, tablets, pills, capsules, lozenges, suppositories, patches, nasal sprays, injectables, implantable sustained release formulations, mucoadhesive films, topical varnishes, lipid complexes, etc.

Pharmaceutical compositions comprising the active agents described herein (e.g., the hydantoins described herein, or one or more tautomers or stereoisomers thereof, or pharmaceutically acceptable salts or solvates of said one or more hydantoins, said one or more stereoisomers, or said one or more tautomers thereof) can be manufactured by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. The pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the one or more active agents into preparations which can be used pharmaceutically. The suitable formulation depends on the route of administration chosen.

In certain embodiments, the active agents described herein are formulated for oral administration. For oral administration, suitable formulations can be readily formulated by combining the one or more active agents with pharmaceutically acceptable carriers suitable for oral administration well known in the art. Such carriers allow the one or more active agents described herein to be formulated as tablets, pills, dragees, caplets, lozenges, gelcaps, capsules, liquids, gels, syrups, pastes, suspensions, and the like, for oral ingestion by a patient to be treated. For oral solid formulations such as, for example, powders, capsules and tablets, suitable excipients may include fillers such as sugars (e.g., lactose, sucrose, mannitol and sorbitol), cellulose preparations (e.g., corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose), synthetic polymers (e.g., polyvinylpyrrolidone (PVP)), granulating agents; and binding agents. If desired, disintegrating agents, such as cross-linked polyvinylpyrrolidone, agar or alginic acid or a salt thereof, such as sodium alginate, may be added. If desired, solid dosage forms may be sugar coated or enteric coated using standard techniques. The preparation of enteric coated particles is described, for example, in U.S. Pat. U.S. Nos. 4,786,505 and 4,853,230.

For administration by inhalation, the one or more active agents are conveniently supplied as an aerosol from pressurised packs or a nebuliser, with the use of a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve for delivering a metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

In various embodiments, the one or more active agents can be formulated into rectal or vaginal compositions such as suppositories or retention enemas, containing, for example, conventional suppository bases, such as cocoa butter or other glycerides. Methods for formulating active agents for rectal or vaginal delivery are well known to those skilled in the art (see, e.g., Allen (2007) Suppositories, Pharmaceutical Press) and typically involve combining the active agents with a suitable base (e.g., hydrophilic (PEG), lipophilic materials such as cocoa butter or Witepsol W45, amphiphilic materials such as Suppocire AP and polyglycolized glyceride, and the like). The base is selected and compounded to obtain a desired melting/delivery profile.

For topical administration, the one or more active agents described herein (e.g., the hydantoins described herein, or one or more tautomers or stereoisomers thereof, or pharmaceutically acceptable salts or solvates of said one or more hydantoins, said one or more stereoisomers, or said one or more tautomers thereof) can be formulated as solutions, gels, ointments, creams, suspensions and the like as is well known in the art.

In certain embodiments, the active agents described herein are formulated for systemic administration (e.g., as an injectable) according to standard methods well known to those skilled in the art. Systemic formulations include, but are not limited to, those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal, or intraperitoneal injection, as well as those designed for transdermal, oral transmucosal, or pulmonary administration. For injection, the active agents described herein may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer, and/or in certain emulsion formulations. The one or more solutions may contain formulatory agents such as suspending, stabilizing, and/or dispersing agents. In certain embodiments, the one or more active agents may be provided in powder form for constitution with a suitable vehicle, e.g., pyrogen-free sterile water, prior to use. For transmucosal administration and/or for passage of the blood-brain barrier, penetrants appropriate to the barrier to be permeated may be used in the formulation. Such penetrants are generally known in the art. Injectable formulations and inhalable formulations are generally provided as a sterile or substantially sterile formulation.

In addition to the formulations described above, the one or more active agents may also be formulated as depot preparations. Such long-acting formulations may be administered by implant (e.g., subcutaneous or intramuscular) or by intramuscular injection. Thus, for example, the one or more active agents may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., as a sparingly soluble salt.

In certain embodiments, the one or more active agents described herein may also be delivered through the skin using conventional transdermal drug delivery systems, i.e., transdermal "patches" wherein the one or more active agents are typically contained within a laminated structure that serves as a drug delivery device to be affixed to the skin. In such a structure, the drug composition is typically contained in a layer, or "reservoir," underlying an upper backing layer. It will be appreciated that the term "reservoir" in this context refers to an amount of "one or more active ingredients" that are ultimately available for delivery to the surface of the skin. Thus, for example, the "reservoir" may include the one or more active ingredients in an adhesive on a backing layer of the patch, or in any of a variety of different matrix formulations known to those skilled in the art. The patch may contain a single reservoir or may contain multiple reservoirs.

In an illustrative embodiment, the reservoir comprises a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to secure the system to the skin during drug delivery. Examples of suitable skin contact adhesive materials include, but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates, polyurethanes, and the like. Alternatively, the drug-containing reservoir and the skin contact adhesive are present as separate and distinct layers, with the adhesive underlying the reservoir which, in this case, may be a polymeric matrix as described above, or may be a liquid or hydrogel reservoir, or may take some other form. The backing layer in these laminates, which serves as the top surface of the device, preferably functions as a primary structural element of the "patch" and provides the device with much of its flexibility. The material selected for the backing layer is preferably substantially impermeable to the one or more active agents and any other materials that are present.

Alternatively, other pharmaceutical delivery systems may be employed. For example, liposomes, emulsions and microemulsions/nanoemulsions are well-known examples of delivery vehicles that can be used to protect and deliver pharmaceutically active compounds. Certain organic solvents such as dimethyl sulfoxide may also be employed, although usually at the cost of increased toxicity.

In certain embodiments, the one or more active agents described herein (e.g., the hydantoins described herein, or one or more tautomers or stereoisomers thereof, or pharmaceutically acceptable salts or solvates of said one or more hydantoins, said one or more stereoisomers, or said one or more tautomers thereof) are formulated in a nanoemulsion. Nanoemulsions include, but are not limited to, oil-in-water (O/W) nanoemulsions and water-in-oil (W/O) nanoemulsions. Nanoemulsions can be defined as emulsions with average droplet diameters ranging from about 20 to about 1000 nm. Typically, the average droplet size is between about 20 nm or 50 nm and about 500 nm. The terms submicron emulsion (SME) and miniemulsion are used synonymously.

Illustrative oil-in-water (O/W) nanoemulsions include, but are not limited to: Surfactant micelles: Micelles composed of small surfactant or detergent molecules (e.g., SDS/PBS/2-propanol); Polymer micelles: Micelles composed of polymer, copolymer, or block copolymer surfactants (e.g., Pluronic L64/PBS/2-propanol); Mixed micelles: Micelles in which more than one surfactant component is present or in which one of the liquid phases (usually an alcohol or fatty acid compound) participates in the formation of the micelle (e.g., octanoic acid/PBS/EtOH); Integral micelles: Mixed micelles in which the one or more active agents serve as an auxiliary surfactant, forming an integral part of the micelle; and Pickering emulsions (solid phase): Emulsions in which the one or more active agents are associated with the exterior of a solid nanoparticle (e.g. polystyrene/PBS/no oil phase nanoparticles).

Illustrative water-in-oil (W/O) nanoemulsions include, but are not limited to: Surfactant micelles: Micelles composed of small surfactant or detergent molecules (e.g., dioctyl sulfosuccinate/PBS/2-propanol, isopropyl myristate/PBS/2-propanol, etc.); Polymer micelles: Micelles composed of polymer, copolymer, or block copolymer surfactants (e.g., PLURONIC® L121/PBS/2-propanol); Mixed micelles: Micelles in which more than one surfactant component is present or in which one of the liquid phases (usually an alcohol or fatty acid compound) is involved in the formation of the micelle (e.g., capric/caprylic diglyceride/PBS/EtOH); Integral micelles: Mixed micelles in which the one or more active agents serve as auxiliary surfactant, forming an integral part of the micelle (e.g., active agent/PBS/polypropylene glycol); and Pickering emulsions (solid phase): Emulsions in which the one or more active agents are associated with the exterior of a solid nanoparticle (e.g., chitosan nanoparticles/no aqueous phase/mineral oil).

As noted above, in certain embodiments, the nanoemulsions comprise one or more surfactants or detergents. In some embodiments, the surfactant is a nonionic detergent (e.g., a polysorbate surfactant, a polyoxyethylene ether, etc.). Surfactants that find use in the present invention include, but are not limited to, surfactants such as the TWEEN®, TRITON®, and TYLOXAPOL® families of compounds.

In certain embodiments, the emulsions further comprise one or more halogen-containing cationic compounds, including, but not limited to, cetylpyridinium chloride. In still other embodiments, the compositions further comprise one or more compounds that increase the interaction ("interaction enhancers") of the composition with microorganisms (e.g., chelating agents such as ethylenediaminetetraacetic acid or ethylenebis(oxyethylenenitrile)tetraacetic acid in a buffer).

In some embodiments, the nanoemulsion further comprises an emulsifying agent to aid in the formation of the emulsion. Emulsifying agents include compounds that aggregate at the oil/water interface to form a continuous membrane-like structure that prevents direct contact between two adjacent droplets. Certain embodiments of the present invention feature oil-in-water emulsion compositions that can be readily diluted with water to a desired concentration without impairing their antipathogenic properties.

In addition to discrete oil droplets dispersed in an aqueous phase, certain oil-in-water emulsions may also contain other lipid structures, such as small lipid vesicles (e.g., lipid spheres often consisting of several substantially concentric lipid bilayers separated from each other by layers of aqueous phase), micelles (e.g., amphiphilic molecules in small clusters of 50-200 molecules arranged such that the polar head groups face outward toward the aqueous phase and the nonpolar tails are sequestered inward away from the aqueous phase), or lamellar phases (lipid dispersions in which each particle consists of parallel amphiphilic bilayers separated by thin films of water).

These lipid structures are formed as a result of hydrophobic forces that draw nonpolar residues (e.g. long hydrocarbon chains) away from water. The above lipid preparations can be generally described as surfactant lipid preparations (SLPs). SLPs are minimally toxic to mucous membranes and are thought to be metabolized within the small intestine (see, e.g., Hamouda et al.,(1998) J.Infect. Disease180:1939).

In certain embodiments, the emulsion comprises a discontinuous oil phase distributed in an aqueous phase, a first component comprising an alcohol and/or glycerol, and a second component comprising a surfactant or a halogen-containing compound. The aqueous phase may comprise any type of aqueous phase including, but not limited to, water (e.g., deionized water, distilled water, tap water) and solutions (e.g., phosphate buffered saline or other buffer systems). The oil phase may comprise any type of oil including, but not limited to, vegetable oils (e.g., soybean oil, avocado oil, linseed oil, coconut oil, cottonseed oil, squalene oil, olive oil, canola oil, corn oil, rapeseed oil, safflower oil, and sunflower oil), animal oils (e.g., fish oil), flavoring oil, water-insoluble vitamins, mineral oil, and motor oil. In certain embodiments, the oil phase comprises 30-90% by vol. of the oil-in-water emulsion (e.g., constitutes 30-90% of the total volume of the final emulsion), more preferably 50-80%. Formulations need not be limited to particular surfactants, however, in certain embodiments, the surfactant is a polysorbate surfactant (e.g., TWEEN 20®, TWEEN 40®, TWEEN 60®, and TWEEN 80®), a pheoxypolyethoxyethanol (e.g., TRITON® X-100, X-301, X-165, X-102, and X-200, and TYLOXAPOL®), or sodium dodecyl sulfate, and the like.

In certain embodiments, a halogen-containing component is present, the nature of the halogen-containing compound, in some embodiments, the halogen-containing compound comprises a chloride salt (e.g., NaCl, KCl, etc.), a cetylpyridinium halide, a cetyltrimethylammonium halide, a cetyldimethylethylammonium halide, a cetyldimethylbenzylammonium halide, a cetyltributylphosphonium halide, dodecyltrimethylammonium halides, tetradecyltrimethylammonium halides, cetylpyridinium chloride, cetyltrimethylammonium chloride, cetylbenzyldimethylammonium chloride, cetylpyridinium bromide, cetryltrimethylammonium bromide, cetyldimethylethylammonium bromide, cetyltributylphosphonium bromide, dodecyltrimethylammonium bromide, tetradecyltrimethylammonium and the like.

In certain embodiments, the emulsion comprises a quaternary ammonium compound. Quaternary ammonium compounds include, but are not limited to, N-alkyldimethyl benzyl ammonium saccharinate, 1,3,5-triazin-1,3,5(2H,4H,6H)-triethanol; 1-decanaminium, N-decyl-N,N-dimethyl-, didecyl dimethyl ammonium chloride (or) chloride; 2-(2-(p-(diisobutyl)cresosoxy)ethoxy)ethyl dimethyl benzyl ammonium chloride; 2-(2-(p-(diisobutyl)phenoxy)ethoxy)ethyl dimethyl benzyl ammonium chloride; 1- or 3-alkyl benzyl-1-(2-hydroxyethyl)-2-imidazolinium chloride; alkyl bis(2-hydroxyethyl)benzyl ammonium chloride; alkyl dimethyl benzyl ammonium chloride; alkyl dimethyl 3,4-dichlorobenzyl ammonium chloride (100% C12); alkyl dimethyl 3,4-dichlorobenzyl ammonium chloride (50% C14, 40% C12, 10% C16); alkyl dimethyl 3,4-dichlorobenzyl ammonium chloride (55% C14, 23% C12, 20% C16); alkyl dimethyl benzyl ammonium chloride; alkyl dimethyl benzyl ammonium chloride (100% C14); alkyl dimethyl benzyl ammonium chloride (100% C16); alkyl dimethyl benzyl ammonium chloride (41% C14, 28% C12); alkyl dimethyl benzyl ammonium chloride (47% C12, 18% C14); alkyl dimethyl benzyl ammonium chloride (55% C16, 20% C14); alkyl dimethyl benzyl ammonium chloride (58% C14, 28% C16); alkyl dimethyl benzyl ammonium chloride (60% C14, 25% C12); alkyl dimethyl benzyl ammonium chloride (61% C11, 23% C14); alkyl dimethyl benzyl ammonium chloride (61% C12, 23% C14); alkyl dimethyl benzyl ammonium chloride (65% C12, 25% C14); alkyl dimethyl benzyl ammonium chloride (67% C12, 24% C14); alkyl dimethyl benzyl ammonium chloride (67% C12, 25% C14); alkyl dimethyl benzyl ammonium chloride (90% C14, 5% C12); alkyl dimethyl benzyl ammonium chloride (93% C14, 4% C12); alkyl dimethyl benzyl ammonium chloride (95% C16, 5% C18); alkyl dimethyl benzyl ammonium chloride (and) didecyl dimethyl ammonium chloride; alkyl dimethyl benzyl ammonium chloride (as in fatty acids); alkyl dimethyl benzyl ammonium chloride (C12-C16); alkyl dimethyl benzyl ammonium chloride (C12-C18); alkyl dimethyl benzyl dialkyl dimethyl ammonium chloride; alkyl dimethyl dimethylbenzyl ammonium chloride; alkyl dimethyl ethyl ammonium bromide (90% C14, 5% C16, 5% C12); alkyl dimethyl ethyl ammonium bromide (mixed alkyl and alkenyl groups as in soybean oil fatty acids); alkyl dimethyl ethylbenzyl ammonium chloride; alkyl dimethyl ethylbenzyl ammonium chloride (60% C14); alkyl dimethyl isopropylbenzyl ammonium chloride (50% C12, 30% C14, 17% C16, 3% C18); alkyl trimethyl ammonium chloride (58% C18, 40% C16, 1% C14, 1% C12); alkyl trimethyl ammonium chloride (90% <c>18, 10% C16); alkyldimethyl(ethylbenzyl) ammonium chloride (C12-18); di-alkyl(C8-10)dimethyl ammonium chlorides; dialkyl dimethyl ammonium chloride; dialkyl dimethyl ammonium chloride; dialkyl dimethyl ammonium chloride; dialkyl methyl benzyl ammonium chloride; didecyl dimethyl ammonium chloride; diisodecyl dimethyl ammonium chloride; dioctyl dimethyl ammonium chloride; dodecyl bis(2-hydroxyethyl) octyl hydrogen ammonium chloride; dodecyl dimethyl benzyl ammonium chloride; dodecylcarbamoyl methyl dimethyl benzyl ammonium chloride; heptadecyl hydroxyethylimidazolinium chloride; hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine; myristalkonium chloride (and) Quat RNIUM 14; N,N-dimethyl-2-hydroxypropylammonium chloride polymer; n-alkyl dimethyl benzyl ammonium chloride; n-alkyl dimethyl ethylbenzyl ammonium chloride; tetradecyl dimethyl benzyl ammonium chloride monohydrate; octyl decyl dimethyl ammonium chloride; octyl dodecyl dimethyl ammonium chloride; octyphenoxyethoxyethyl dimethyl benzyl ammonium chloride; oxydiethylenebis(alkyl dimethyl ammonium chloride); quaternary ammonium compounds, dicoco alkyldimethyl, chloride; trimethoxysilyl propyl dimethyl octadecyl ammonium chloride; trimethoxysilyl quats, trimethyl dodecylbenzyl ammonium chloride; n-dodecyl dimethyl ethylbenzyl ammonium chloride; n-hexadecyl dimethyl benzyl ammonium chloride; n-tetradecyl dimethyl benzyl ammonium chloride; n-tetradecyl dimethyl ethylbenzyl ammonium chloride; and n-octadecyl dimethyl benzyl ammonium chloride.

Nanoemulsion formulations and methods for preparing them are well known to those skilled in the art and are described, for example, in U.S. Patent Nos. 7,476,393; 7,468,402; 7,314,624; 6,998,426; 6,902,737; 6,689,371; 6,541,018; 6,464,990; 6,461,625; 6,419,946; 6,413,527; 6,375,960; 6,335,022; 6,274,150; 6,120,778; 6,039,936; 5,925,341; 5,753,241; 5,698,219, and 5,152,923 and in Fanunet al. (2009) Microemulsions: Properties and Applications (Surfactant Science), CRC Press, Boca Raton, Fl.

In certain embodiments, one or more active agents described herein may be provided as a "concentrate," for example, in a storage container (e.g., in a premeasured volume) ready for dilution, or in a soluble capsule ready for addition to a volume of water, alcohol, hydrogen peroxide, or other diluent.

Extended-release (sustained-release) formulations.

In certain embodiments, "extended release" formulations of the one or more active agents described herein are contemplated (e.g., the hydantoins described herein, or one or more tautomers or stereoisomers thereof, or pharmaceutically acceptable salts or solvates of said one or more hydantoins, said one or more stereoisomers, or said one or more tautomers thereof). In various embodiments, such extended release formulations are designed to avoid the high peak plasma levels of conventional immediate release intravenous and oral dosage forms.

Illustrative sustained release formulations include, for example, semipermeable matrices of solid polymers containing the therapeutic agent. Various uses of sustained release materials have been established and are well known to those skilled in the art. Sustained release capsules may, depending on their chemical nature, release compounds for a few weeks to over 100 days. Depending on the chemical nature and biological stability of the therapeutic reagent, additional strategies for stabilization may be employed.

In certain embodiments, such "extended release" formulations utilize the mucosa and can independently control disintegration (or erosion) of the tablet and/or dissolution and release of the drug from the tablet over time to provide a safer delivery profile. In certain embodiments, oral formulations of the one or more active agents described herein (e.g., the hydantoins described herein, or one or more tautomers or stereoisomers thereof, or pharmaceutically acceptable salts or solvates of said one or more hydantoins, said one or more stereoisomers, or said one or more tautomers thereof) provide single, repetitive doses that include a defined amount of the active agent that is delivered over a defined period of time.

An exemplary sustained release formulation is a substantially homogeneous composition comprising from about 0.01% to about 99% w/w, or from about 0.1% to about 95%, or about 0.1%, or about 1%, or about 2%, or about 5%, or about 10%, or about 15%, or from about 20% to about 80%, or about 90%, or about 95%, or about 97%, or about 98%, or about 99% of the one or more active ingredients (e.g., the hydantoins described herein, or one or more tautomers or stereoisomers thereof, or pharmaceutically acceptable salts or solvates of said one or more hydantoins, said one or more stereoisomers, or said one or more tautomers thereof) and one or more mucoadhesives. (also referred to herein as "bioadhesives") that provide adherence to the subject's (patient's) target mucosa and which may further comprise one or more of the following: one or more binders that provide attachment of the excipients into a single tablet; one or more hydrogel-forming excipients; one or more bulking agents; one or more lubricants; one or more glidants; one or more solubilizers; one or more surfactants; one or more flavors; one or more disintegrants; one or more buffering excipients; one or more coatings; one or more controlled release modifiers; and one or more excipients and factors that modify and control the time and kinetics of dissolution or disintegration of the drug or that protect the active drug from degradation.

In various embodiments, a sustained-release dosage form for oral transmucosal delivery may be solid or non-solid. In an illustrative embodiment, the dosage form is a solid that converts to a hydrogel upon contact with saliva.

Suitable excipients include, but are not limited to, substances added to formulations that are required to produce a commercial product and may include, but are not limited to: bulking agents, binders, surfactants, bioadhesives, lubricants, disintegrants, stabilizers, solubilizers, glidants, and additives or factors which affect dissolution or disintegration time. Suitable excipients are not limited to the above, and other nontoxic pharmaceutically acceptable carriers suitable for use in oral formulations can be found in Remington's Pharmaceutical Sciences, 17th Edition, 1985.

In certain embodiments, extended release formulations of the one or more active agents described herein for oral transmucosal drug delivery include at least one bioadhesive agent (mucoadhesive) or a mixture of several bioadhesives to promote adhesion to the oral mucosa during drug delivery. In addition, bioadhesive agents may also be effective in controlling the erosion time of the dosage form and/or the dissolution kinetics of the drug over time when the dosage form is wetted. Such mucoadhesive drug delivery systems are highly beneficial as they can prolong the residence time of the drug at the absorption site and increase the bioavailability of the drug. Mucoadhesive polymers that form hydrogels are typically hydrophilic and swellable, and contain numerous hydrogen bond-forming groups, such as hydroxyl, carboxyl, or amine, that promote adhesion. When used in dry form, they attract water from the mucosal surface and swell, leading to polymer/mucus interaction through hydrogen bonding, electrostatic, hydrophobic or van der Waals interaction.

Illustrative suitable mucoadhesive or bioadhesive materials include, but are not limited to, natural, synthetic or biological polymers, lipids, phospholipids and the like. Examples of natural and/or synthetic polymers include cellulosic derivatives (such as methylcellulose, carboxymethylcellulose, hydroxyethylcellulose, hydroxyethylmethylcellulose, etc.), natural gums (such as guar gum, xanthan gum, locust bean gum, karaya gum, veegum gum, etc.), polyacrylates (such as CARBOPOL®, polycarbophil, etc.), alginates, polymers containing thiols, POLYOX® ethylenes, polyethylene glycols (PEG) of all molecular weights (preferably between 1,000 and 40,000 Da, of any chemistry, linear or branched), dextrans of all molecular weights (preferably between 1,000 and 40,000 Da from any source), block copolymers, such as those prepared by combinations of lactic and glycolic acid (PLA, PGA, PLGA of various viscosities, etc.), molecular weights and ratios of lactic glycolic acid), polyethylene glycol-polypropylene glycol block copolymers of any number and combination of repeating units (such as Pl Ur On ICS®, TEKTRONIX® or GENAPOL® block copolymers), combination of the above copolymers whether physically or chemically linked units (e.g., PEG-PLA or PEG-PLGA copolymer blends). Preferably, the bioadhesive excipient is selected from the group consisting of polyethylene glycols, POLYOX® ethylenes, polyacrylic acid polymers such as CARBOPOL® (such as CARBOPOL® 71G, 934P, 971P, 974P and the like) and polycarbophils (such as NOVEON® AA-1, NOVEON® CA-1, NOVEON® CA-2 and the like), cellulose and its derivatives, and most preferably is polyethylene glycol, carbopol and/or a cellulosic derivative or a combination thereof.

In certain embodiments, the mucoadhesive/bioadhesive excipient is typically present at 1-50% w/w, preferably 1-40% w/w or most preferably 5-30% w/w. A particular formulation may contain one or more different bioadhesives in any combination.

In certain embodiments, formulations for oral transmucosal drug delivery also include a binder or a mixture of two or more binders that facilitate the binding of the excipients into a single dosage form. Illustrative binders include binders selected from the group consisting of cellulosic derivatives (such as methylcellulose, carboxymethylcellulose, hydroxyethylcellulose, hydroxyethylmethylcellulose, etc.), polyacrylates (such as CARBOPOL®, polycarbophil, etc.), POVIDONE® (all grades), POLYOX®® of any molecular weight or grade, irradiated or not, starch, polyvinylpyrrolidone (PVP), AVICEL®, and the like. In certain embodiments, the binder is typically present at 0.5-60% w/w, preferably 1-30% w/w, and most preferably 1.5-15% w/w.

In certain embodiments, the formulations also include at least one hydrogel-forming excipient. Illustrative hydrogel-forming excipients include, but are not limited to, those selected from the group consisting of polyethylene glycols and other polymers having an ethylene glycol backbone, whether cross-linked homopolymers or heteropolymers, block copolymers using ethylene glycol units, such as POLYOX® ethylene homopolymers (such as POLYOX® N10/MW = 100,000 POLYOX®-80/MW = 200,000; POLYOX® 1105/MW = 900,000; POLYOX®-301/MW = 4,000,000; POLYOX®-303/MW = 7,000,000, POLYOX® WSR-N-60K, all of which are trade names of Union Carbide), hydroxypropyl methylcellulose (HPMC) of all molecular weights and grades (such as METOLOSE® 1000, POLYOX ... 90SH50000, METo Lo Se® 90SH30000, all of which are trade names of Shin-Etsu Chemical company), poloxamers (such as LUTROL® F-68, LUTROL® F-127, F-105,etc.,all trade names of Ba Sf Chemicals), GEnA p OL®, polyethylene glycols (PEGs, such as PEG-1500, PEG-3500, PEG-4000, PEG-6000, PEG-8000, PEG-12000, PEG-20,000,etc.), natural gums (xanthan gum, locust bean gum, etc.) and cellulose derivatives (HC, HMC, HMPC, HPC, CP, CMC), free or crosslinked polyacrylic acid-based polymers and combinations thereof, biodegradable polymers such as polylactic acids, carboxylic acids, polyglycolic and any combination thereof, whether a physical mixture or cross-linked. In certain embodiments, the hydrogel components may be cross-linked. The one or more hydrogel-forming excipients are typically present at 0.1-70% w/w, preferably 1-50% w/w or most preferably 1-30% w/w.

In certain embodiments, the formulations may also include at least one controlled release modifier which is a substance that upon hydration of the dosage form will preferentially adhere to the drug molecules and thus reduce the rate of their diffusion from the oral dosage form. Such excipients may also reduce the rate of water absorption by the formulation and thus allow for a more prolonged dissolution and release of the drug from the tablet. In general, the one or more excipients selected are lipophilic and are capable of naturally forming complexes with hydrophobic or lipophilic drugs. The degree of association of the release modifier and the drug may be varied by altering the modifier-drug ratio in the formulation. Furthermore, such interaction may be suitably enhanced by appropriate combination of the release modifier with the active drug in the manufacturing process. Alternatively, the controlled release modifier may be a charged polymer, either synthetic or biopolymeric, that carries a net charge, either positive or negative, and that is capable of binding to the active ingredient through electrostatic interactions, thereby modifying both its diffusion through the tablet and the kinetics of its penetration through the mucosal surface. Similar to the other compounds mentioned above, said interaction is reversible and does not involve permanent chemical bonds with the active ingredient. In certain embodiments, the controlled release modifier may typically be present at 0-80% w/w, preferably 1-20% w/w, most preferably 1-10% w/w.

In various embodiments, the extended-release formulations may also include other conventional components necessary for the development of oral dosage forms, which are known to those skilled in the art. These components may include one or more bulking agents (such as lactose USP, Starch 1500, mannitol, sorbitol, malitol or other non-reducing sugars; microcrystalline cellulose (e.g., AVICEL®), dehydrated dibasic calcium phosphate, sucrose, and mixtures thereof), at least one or more solubilizing agents (such as cyclodextrins, pH adjusters, salts and buffers, surfactants, fatty acids, phospholipids, fatty acid metals, etc.), metal salts and organic (such as acetate, citrate, tartrate, etc.) or inorganic (phosphate, carbonate, bicarbonate, borate, sulfate, sulfite, bisulfite, metabisulfite, chloride, etc.) buffers, metal salts such as sodium, potassium, calcium, magnesium, etc.), at least one lubricant (such as stearic acid and divalent cations, such as magnesium stearate, potassium stearate, etc.), and/or glycerin. calcium, etc., talc, glycerol monostearate and the like), one or more glidants (such as colloidal silicon dioxide, precipitated silicon dioxide, fumed silica (CABO-SIL® M-5P, trademark of Cabot Corporation), stearowet and sterotex, silicas (such as SILOID® and SILOX® silicas, trademarks of Grace Davison Products, Aerosil, trademark of Degussa Pharma), higher fatty acids, the metal salts thereof, hydrogenated vegetable oils and the like), flavorings or sweetening matters and coloring matters (such as aspartame, mannitol, lactose, sucrose, other artificial sweeteners; Ferric oxides and FD&C lakes), additives to help stabilize the drug substance against chemical or physical degradation (such as antioxidants, antihydrolytic agents, aggregation blockers, etc. Antioxidants may include BHT, BHA, vitamins, citric acid, EDTA, sodium bisulfate, sodium metabisulfate, thiourea, methionine, surfactants, amino acids such as arginine, glycine, histidine, methionine salts, pH adjusters, chelating agents, and buffers in dry or solution form), one or more excipients that may affect the disintegration kinetics of the tablet and the release of the drug from the tablet and therefore the pharmacokinetics (disintegrants such as those known to those skilled in the art and may be selected from the group consisting of starch, carboxymethylcellulose type or cross-linked polyvinylpyrrolidone (such as povidone, PVP-XL), alginates, disintegrants such as carboxymethylcellulose, or cross-linked polyvinylpyrrolidone (such as povidone, PVP-XL), ... cellulose base (such as purified cellulose, methylcellulose, cross-linked sodium carboxymethylcellulose (Ac-Di-Sol) and carboxymethylcellulose), low substituted hydroxypropyl ethers of cellulose, microcrystalline cellulose (such as AVICEL®), ion exchange resins (such as AMBRELITE® IPR 88), gums (such as agar, locust bean, karaya, pectin and tragacanth), guar gums, karaya gum, chitin and chitosan, smecta, gellan gum, isapghula shell, polacrilin potassium (Tulsion 339), gas-evolving disintegrants (such as citric acid and tartaric acid together with sodium bicarbonate, sodium carbonate, potassium bicarbonate or calcium carbonate), sodium starch glycolate (such as EXPLOTAB® and PRIMOGEL®), DC starch and the like, at least one biodegradable polymer of any type useful for the sustained release of drugs. Exemplary polymeric compositions include, but are not limited to, polyanhydrides and copolymers of lactic acid and glycolic acid, poly(dl-lactide-coglycolide) (PLGA), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), polyorthoesters, proteins, and polysaccharides.

In certain embodiments, the one or more active agents can be chemically modified to significantly alter plasma pharmacokinetics. This can be achieved, for example, by conjugation with polyethylene glycol (PEG), including site-specific PEGylation. PEGylation, which can improve drug performance by optimizing pharmacokinetics, decreasing immunogenicity and dosing frequency.

Also provided are methods for preparing a formulation of the one or more active agents described herein (e.g., the hydantoins described herein, or one or more tautomers or stereoisomers thereof, or pharmaceutically acceptable salts or solvates of said one or more hydantoins, said one or more stereoisomers, or said one or more tautomers thereof) for gastrointestinal or oral transmucosal delivery. One method includes the steps of grinding the powder, mixing the dry powder, and tableting by direct compression. Alternatively, a wet granulation process may be used. Such a method (such as a high shear granulation process) involves mixing the active ingredient and possibly some excipients in a blender. The binder may be one of the excipients added in a dry blend state or dissolved in the fluid used for granulation. The granulating solution or suspension is added to the dry powders in the blender and mixed until the desired characteristics are achieved. This usually produces a granule that will have characteristics suitable for producing dosage forms with suitable dissolution time, content uniformity, and other physical characteristics. Following the wet granulation step, the product is most often dried and/or then ground after drying to obtain a significant percentage of the product within a desired size range. Occasionally, the product is dried after wet sizing using a device such as an oscillating granulator or mill. The dried granulation can then be processed to obtain an acceptable size range by first screening with a screening device and then grinding the oversized particles.

Furthermore, the formulation may be manufactured by alternative granulation processes, all known to those skilled in the art, such as fluid bed spray granulation, extrusion and spheronization or rotor fluid bed granulation.

Furthermore, the tablet dosage form of the one or more active agents described herein may be prepared by coating the primary tablet manufactured as described above with suitable coatings known in the art. Such coatings are intended to protect the active cores against damage (abrasion, breakage, dust formation) against the influences to which the cores are exposed during transport and storage (atmospheric humidity, temperature fluctuations) and naturally these film coatings may also be colored. The sealing effect of the films against water vapor is expressed by the water vapor permeability. The coating may be accomplished by one of the available processes such as Wurster coating, dry coating, film coating, fluid bed coating, pan coating, etc. Typical coating materials include polyvinylpyrrolidone (PVP), polyvinylpyrrolidone-vinyl acetate copolymer (PVPVA), polyvinyl alcohol (PVA), polyvinyl alcohol/polyethylene glycol copolymer (PVA/PEG), cellulose acetate phthalate, ethylcellulose, gellan gum, maltodextrin, methacrylates, methylcellulose, hydroxypropyl methylcellulose (HPMC of all grades and molecular weights), carrageenan, shellac and the like.

In certain embodiments, the tablet core comprising the one or more active agents described herein may be coated with a bioadhesive and/or pH-resistant material to allow a material, such as those defined above, to enhance bioadhesion of the tablet in the sublingual cavity.

In certain embodiments, the one or more active agents described herein (e.g., the hydantoins described herein, or one or more tautomers or stereoisomers thereof, or pharmaceutically acceptable salts or solvates of said one or more hydantoins, said one or more stereoisomers, or said one or more tautomers thereof) are formulated as inclusion complexes. Although not limited to cyclodextrin inclusion complexes, it should be noted that cyclodextrin is the most frequently used agent to form pharmaceutical inclusion complexes. Cyclodextrins (CDs) are cyclic oligomers of glucose, typically containing 6, 7, or 8 glucose monomers linked by α-1,4 linkages. These oligomers are commonly referred to as α-CDs, p-CDs, and y-CDs, respectively. Higher oligomers containing up to 12 glucose monomers are known and are contemplated in the formulations described herein. Functionalized cyclodextrin inclusion complexes are also contemplated. Illustrative, but not limiting, functionalized cyclodextrins include, but are not limited to, sulfonates, sulfonates and sulfinates, or disulfonates of hydroxybutenyl cyclodextrin; sulfonates, sulfonates and sulfinates, or disulfonates of mixed ethers of cyclodextrins where at least one of the ether substituents is hydroxybutenyl cyclodextrin. Illustrative cyclodextrins include a polysaccharide ether comprising at least one 2-hydroxybutenyl substituent, wherein the at least one hydroxybutenyl substituent is sulfonated and sulfinated, or disulfonated, and an alkyl polyglucoside ether comprising at least one 2-hydroxybutenyl substituent, wherein the at least one hydroxybutenyl substituent is sulfonated and sulfinated, or disulfonated. In various embodiments, inclusion complexes formed between sulfonated hydroxybutenyl cyclodextrins and one or more of the one or more active agents described herein are contemplated. Methods for preparing cyclodextrins and cyclodextrin inclusion complexes are found, for example, in U.S. Patent Publication No.: 2004/0054164 and references cited therein, and in U.S. Patent Publication No.: 2011/0218173 and references cited therein.

Pharmacokinetics (PK) and formulation attributes

An advantage of the oral (GI or transmucosal) extended-release (controlled) formulations described herein is that they can maintain the plasma drug concentration within a specific therapeutic window for a longer period than with immediate-release formulations, whether solid dosage forms or liquid-based dosage forms. The high peak plasma levels typically observed for such conventional immediate-release formulations will be mitigated by the prolonged release of the drug over 1 to 12 hours or more. In addition, a rapid decline in plasma levels will be avoided, as the drug will continuously pass from the oral cavity into the bloodstream during the dissolution time of the tablet, thus providing plasma pharmacokinetics with a more stable plateau. In addition, the dosage forms described herein may improve treatment safety by minimizing potentially harmful side effects due to reduced peaks and valleys in plasma drug pharmacokinetics, which compromise treatment safety.

In various embodiments, the oral transmucosal formulations of the one or more active agents described herein are designed to avoid the high peak plasma levels of conventional intravenous immediate release oral dosage forms by utilizing the mucosa and independently controlling both tablet disintegration (or erosion) and dissolution and release of the drug from the tablet over time to provide a safer delivery profile. The oral formulations described herein provide single, repetitive doses that include a defined amount of the active agent.

An advantage of the bioadhesive oral transmucosal formulations described herein is that they exhibit highly consistent bioavailability and can maintain plasma drug concentration within a specific therapeutic window with significantly lower variability for a longer period than currently available dosage forms, whether solid dosage forms or IV dosage forms. In addition, a rapid decline in plasma levels is avoided as the drug continuously crosses from the oral cavity or GI tract into the bloodstream for the tablet dissolution time or longer, thereby providing the plasma pharmacokinetics with a prolonged plateau phase compared to conventional immediate-release oral dosage forms. Furthermore, the dosage forms described herein may improve treatment safety by minimizing potentially deleterious side effects due to the relative reduction of peaks and valleys in plasma drug pharmacokinetics, which compromise treatment safety and is typical of currently available dosage forms.

In various embodiments, the bioadhesive formulations described herein can be designed to manipulate and control the pharmacokinetic profile of the one or more active agents described herein. As such, the formulations can be tuned to achieve "slow" disintegration times (and erosion kinetic profiles) and slow release of the drug and thus allow for very long pharmacokinetic profiles that provide sustained pharmacokinetic action. Although such formulations can be designed to provide rapid onset, their primary goal is to allow for sustained PK and effect of the drug while maintaining the other performance attributes of the tablet, such as bioadhesion, reproducibility of action, attenuated Cmax, etc.

The performance and attributes of the bioadhesive transmucosal formulations of this invention are independent of the manufacturing process. Various conventional, well-established processes known in the art can be used to manufacture the formulations of the present invention (such as wet and dry granulation, direct compression, etc.) without affecting the physicochemical properties of the dosage form or the in vivo performance.

An illustrative mathematical relationship demonstrating the prolonged plateau phase of measured blood plasma levels of the one or more active agents described herein following administration of the dosage forms of the invention is the term "optimal therapeutic targeting ratio" or "OTTR", which represents the average time that the drug is present at therapeutic levels, defined as the time that the plasma concentration of the drug remains above 50% of the Cmax normalized by the elimination half-life of the drug multiplied by the ratio of the Cmax obtained in the dosage form of interest to the normalized Cmax following IV administration of equivalent doses. In certain embodiments, the OTTR can be calculated by the formula:

OTTR=(CIVmax/Cmax) x (Dose/IVDose) (Time above 50% of Cmax)/(Terminal elimination half-life of the drug).

In certain embodiments, the OTTR is about 15, or greater than about 20, or greater than about 25, or greater than about 30, or greater than about 40, or greater than about 50.

Administration

In certain embodiments, one or more active agents described herein (e.g., the hydantoins described herein, or one or more tautomers or stereoisomers thereof, or pharmaceutically acceptable salts or solvates of said one or more hydantoins, said one or more stereoisomers, or said one or more tautomers thereof) are administered to a mammal in need thereof, for example, a mammal at risk for or suffering from a pathology characterized by abnormal processing of amyloid precursor proteins, a mammal at risk for progression from MCI to Alzheimer's disease, etc. In certain embodiments, the one or more active agents are administered to prevent or delay the onset of a pre-Alzheimer's cognitive dysfunction, and/or to ameliorate one or more symptoms of a pre-Alzheimer's cognitive dysfunction, and/or to prevent or delay the progression of a pre-Alzheimer's cognitive condition or dysfunction to Alzheimer's disease, and/or to promote the processing of amyloid precursor protein (APP) by a non-amyloidogenic pathway. In certain embodiments, the one or more active agents are administered for the treatment of early-stage, mid-stage, or late-stage Alzheimer's disease, e.g., to reduce the severity of the disease and/or ameliorate one or more symptoms of the disease and/or slow the progression of the disease.

In various embodiments, the one or more active agents described herein (e.g., the hydantoins described herein, or one or more tautomers or stereoisomers thereof, or pharmaceutically acceptable salts or solvates of said one or more hydantoins, said one or more stereoisomers, or said one or more tautomers thereof) can be administered by any of several routes. Thus, for example, they can be administered orally, parenterally, (intravenous (IV), intramuscular (IM), depo-IM, subcutaneous (SQ), and depo-SQ), sublingually, intranasally (inhalation), intrathecally, transdermally (e.g., via a transdermal patch), topically, ionophorically, or rectally. Typically, the dosage form is selected to facilitate delivery to the brain (e.g., passage through the blood-brain barrier). In this context, it should be noted that the compounds described herein are readily delivered to the brain. Pharmaceutical forms known to those skilled in the art are suitable for delivery of the compound.

In various embodiments, the one or more active agents are administered in an amount/dosage regimen sufficient to exert a prophylactic and/or therapeutically useful effect in the absence of undesirable side effects in the treated subject (or with the presence of acceptable levels and/or types of side effects). The specific amount/dosage regimen will vary depending on the weight, sex, age, and health of the individual; the formulation, biochemical nature, bioactivity, bioavailability, and side effects of the particular compound.

In certain embodiments, the therapeutically or prophylactically effective amount can be determined empirically by testing the one or more agents in known in vitro and in vivo model systems for the disorder being treated. A therapeutically or prophylactically effective dose can be determined by first administering a low dose and then gradually increasing to a dose that achieves the desired effect with minimal or no unwanted side effects.

In certain embodiments, when administered orally, an administered amount of the one or more agents described herein effective to prevent or delay the onset of a pre-Alzheimer's cognitive dysfunction, and/or to improve one or more symptoms of a pre-Alzheimer's cognitive dysfunction, and/or to prevent or delay the progression of a pre-Alzheimer's cognitive condition or dysfunction to Alzheimer's disease, and/or to promote the processing of amyloid precursor protein (APP) by a non-amyloidogenic pathway, and/or to treat or prevent AD, ranges from about 0.1 mg/day to about 500 mg/day or about 1,000 mg/day, or from about 0.1 mg/day to about 200 mg/day, e.g., from about 1 mg/day to about 100 mg/day, e.g., from about 5 mg/day to about 50 mg/day. In some embodiments, the subject is administered the compound at a dose of about 0.05 to about 0.50 mg/kg, e.g., about 0.05 mg/kg, 0.10 mg/kg, 0.20 mg/kg, 0.33 mg/kg, 0.50 mg/kg. It is understood that while a patient may be started on one dose, that dose may be varied (increased or decreased, as appropriate) over time as the patient's condition changes. Depending on outcome assessments, higher doses may be used. For example, in certain embodiments, up to 1000 mg/day can be administered, e.g., 5 mg/day, 10 mg/day, 25 mg/day, 50 mg/day, 100 mg/day, 200 mg/day, 300 mg/day, 400 mg/day, 500 mg/day, 600 mg/day, 700 mg/day, 800 mg/day, 900 mg/day, or 1000 mg/day.

In various embodiments, the one or more active agents described herein can be administered parenterally, for example, via IV, IM, depo-IM, SC, or depo-SC. In certain embodiments, when administered parenterally, a therapeutically effective amount of about 0.5 to about 100 mg/day, preferably about 5 to about 50 mg per day, can be delivered. When a depot formulation is used for injection once a month or once every two weeks, the dosage in certain embodiments can be about 0.5 mg/day to about 50 mg/day, or a monthly dose of about 15 mg to about 1,500 mg. In part because of forgetfulness in patients with Alzheimer's disease, it is preferred that the parenteral dosage form be a depot formulation.

In various embodiments, the one or more active agents described herein can be administered sublingually. In some embodiments, when administered sublingually, the compounds and/or analogs thereof can be administered one to four times per day in the amounts described above for IM administration.

In various embodiments, the one or more active agents described herein may be administered intranasally. When administered by this route, appropriate dosage forms are a nasal spray or a dry powder, as known to those skilled in the art. In certain embodiments, the dose of the compound and/or analogue thereof for intranasal administration is the amount described above for IM administration.

In various embodiments, the one or more active agents described herein may be administered intrathecally. When administered by this route, the appropriate pharmaceutical form may be a parenteral pharmaceutical form as known to those skilled in the art. In certain embodiments, the dose of the compound and/or analogue thereof for intrathecal administration is the amount described above for IM administration.

In certain embodiments, the one or more active agents described herein can be administered topically. When administered by this route, a suitable pharmaceutical form is a cream, ointment, or patch. When administered topically, the dosage is from about 1.0 mg/day to about 200 mg/day. Because the amount that a patch can deliver is limited, two or more patches may be used. The number and size of the patch is not important as long as a therapeutically effective amount of compound is delivered, as is known to those skilled in the art. The compound can be administered rectally via suppositories as is known to those skilled in the art. In certain embodiments, when administered via suppository, the therapeutically effective amount is from about 1.0 mg to about 500 mg.

In various embodiments, the one or more active agents described herein may be administered via implants as known to those skilled in the art. When the compound is administered via implant, the therapeutically effective amount is the amount described above for depot administration.

In various embodiments, the one or more active agents described herein may be included in single or multiple dose containers. The one or more included agents may be provided in kits, for example, including components that may be assembled for use. For example, an active agent in lyophilized form and a suitable diluent may be provided as separate components for combination prior to use. A kit may include an active agent and a second therapeutic agent for co-administration. The active agent and the second therapeutic agent may be provided as separate components. A kit may include a plurality of containers, each container containing one or more unit doses of the compounds. The containers are preferably adapted to the desired mode of administration, including, but not limited to, tablets, gel capsules, sustained release capsules, and the like for oral administration; depot products, prefilled syringes, ampoules, vials, and the like for parenteral administration; and patches, medicated pads, creams, and the like for topical administration, for example, as described herein.

In various embodiments, the dosage forms may be administered to the subject 1, 2, 3, or 4 times per day. In certain embodiments, it is preferred that the compound be administered three or fewer times, more preferably once or twice per day. In certain embodiments, it is preferred that the one or more agents be administered in an oral dosage form.

It should be apparent to one skilled in the art that the exact dosage and frequency of administration will depend upon the particular condition being treated, the severity of the condition being treated, the age, weight, general physical condition of the particular patient, and other medication the individual may be taking, as is well known to administering physicians skilled in the art.

While the compositions and methods are described herein with respect to use in humans, they are also suitable for use in animals, e.g., veterinary use. Thus, certain organisms (subjects) contemplated herein include, but are not limited to, humans, non-human primates, canids, equines, felines, porcines, ungulates, large-bodied mammals, and the like.

The above formulations and methods of administration are intended to be illustrative and not limiting. It will be appreciated that, using the teachings provided herein, other suitable formulations and modes of administration may be readily devised.

Combined therapies

In certain embodiments, the one or more active agents described herein (e.g., the hydantoins described herein, or one or more tautomers or stereoisomers thereof, or pharmaceutically acceptable salts or solvates of said one or more hydantoins, said one or more stereoisomers, or said one or more tautomers thereof) can be used in conjunction with other therapeutic agents or approaches used to treat or prevent diseases characterized by amyloid deposits in the brain, including MCI and/or AD. Accordingly, in certain embodiments, a pharmaceutical composition comprising at least one active agent described herein (e.g., a hydantoin described herein, or a tautomer or stereoisomer thereof, or pharmaceutically acceptable salts or solvates of said hydantoin, said stereoisomer, or said tautomer thereof) together with at least one additional therapeutic agent, and a pharmaceutically acceptable carrier or diluent is contemplated. In certain embodiments, a therapeutic or prophylactic method is contemplated that comprises administering at least the active agent described herein together with at least one additional therapeutic agent.

In certain embodiments, non-limiting examples of additional therapeutic agents include, but are not limited to, disulfiram and/or analogs thereof, honokiol and/or analogs thereof, tropisetron and/or analogs thereof, nimetazepam and/or analogs thereof (see, e.g., USSN 13/213,960 (U.S. Patent Publication No.: US-2012-0071468-A1), and PCT/US2011/048472 (PCT Publication No.: WO 2012/024616)), tropinol esters and/or related esters and/or analogs thereof (see, e.g., USSN 61/514,381), TrkA kinase inhibitors (e.g., <a>D<d>N-1351) and/or analogs thereof. (see, e.g., USSN 61/525,076), D2 receptor agonists and alpha 1-adrenergic receptor antagonists, and APP-specific BACE inhibitors (ASBI) as described and/or claimed in USSN 61/728,688, filed Tuesday, November 20, 2012.

Non-limiting examples of additional therapeutic agents include drugs selected from the group consisting of: (a) drugs useful for treating Alzheimer's disease and/or drugs useful for treating one or more symptoms of Alzheimer's disease, (b) drugs useful for inhibiting the synthesis of Ap, and (c) drugs useful for treating neurodegenerative diseases. Additional non-limiting examples of additional therapeutic agents for use in conjunction with the compounds (e.g., hydantoins) described herein include drugs useful for treating, preventing, delaying the onset, ameliorating any pathology associated with Ap and/or a symptom thereof. Non-limiting examples of pathologies associated with AP include: Alzheimer's disease, Down syndrome, Parkinson's disease, memory loss, memory loss associated with Alzheimer's disease, memory loss associated with Parkinson's disease, attention deficit symptoms, attention deficit symptoms associated with Alzheimer's disease, Parkinson's disease and/or Down syndrome, dementia, stroke, microgliosis and brain inflammation, pre-senile dementia, senile dementia, dementia associated with Alzheimer's disease, Parkinson's disease and/or Down syndrome, progressive supranuclear palsy, cortical basal degeneration, neurodegeneration, olfactory impairment, olfactory impairment associated with Alzheimer's disease, Parkinson's disease and/or Down syndrome, p-amyloid angiopathy, cerebral amyloid angiopathy, hereditary cerebral hemorrhage, mild cognitive impairment ("MCI"), glaucoma, amyloidosis, type II diabetes, complications of hemodialysis (from p2 microglobulins and complications arising therefrom). in hemodialysis patients), scrapie, bovine spongiform encephalitis, traumatic brain injury ("TBI"), and Creutzfeld-Jakob disease, comprising administering to said patient at least one hydantoin compound described herein, or a tautomer or isomer thereof; or a pharmaceutically acceptable salt or solvate of said compound or said tautomer, in an amount effective to inhibit said pathology(s).

In certain embodiments, such additional therapeutic agents include, but are not limited to, acetylcholinesterase inhibitors (including, without limitation, for example, (-)-phenserine enantiomer, tacrine, ipidacrine, galantamine, donepezil, icopezil, zanapezil, rivastigmine, huperzine A, phenserine, physostigmine, neostigmine, pyridostigmine, ambenonium, demarcarium, edrophonium, dadostigil, and ungeremine); NMDA receptor antagonists (including, without limitation, for example, memantine); muscarinic receptor agonists (including, without limitation, for example, talsaclidine, AF-102B, AF-267B (NGX-267)); nicotinic receptor agonists (including, without limitation, for example, ispronicline (AZD-3480)); beta-secretase inhibitors (including, but not limited to, for example, thiazolidinediones, including rosiglitazone and pioglitazone); gamma-secretase inhibitors (including, but not limited to, for example, semagacestat (LY-450139), Mk-0752, E-2012, BMS-708163, PF-3084014, begacestat (GSI-953), and NIC5-15); Ap aggregation inhibitors (including, but not limited to, for example, clioquinol (PBT1), PBT2, trampiprosate (homotaurine), scyllo-inositol (also known as scyllo-cyclohexanehexol, AZD-103, and ELND-005), passive immunotherapy with Ap fragments (including, but not limited to, for example, bapineuzemab), and epigallocatechin-3-gallate (EGCg)); anti-inflammatory agents such as cyclooxygenase II inhibitors; antioxidants such as vitamin E and ginkgolides; immunological approaches, such as, for example, immunization with Ap peptide or administration of anti-Ap peptide antibodies; statins; and direct or indirect neurotrophic agents such as Cerebrolysin™, AIT-082 (Emilieu, 2000,Arch. Neurol.57:454), netrin (Luorenco (2009)Cell Death Differ.,16:655-663), netrin mimetics, NGF, NGF mimetics, BDNF and other future neurotrophic agents, agents that promote neurogenesis, for example, stem cell therapy. Additional pharmacological agents useful in the treatment or prevention of diseases characterized by amyloid deposits in the brain, including MCI and/or AD, are described, for example, in Mangialasche, et al. (2010) Lancet Neurol., 9:702-716.

In certain embodiments, additional non-limiting examples of additional therapeutic agents for use in conjunction with compounds described herein include: muscarinic antagonists (e.g., cholinesterase inhibitors (e.g., acetylcholine and/or butyrylcholinesterase inhibitors such as donepezil (Aricept®), galantamine (Razadyne®), and rivastigimine (Exelon®); N-methyl-D-aspartate receptor antagonists (e.g., NAMENDA® (memantine HCl); combinations of cholinesterase inhibitors and N-methyl-D-aspartate receptor antagonists; gamma secretase modulators; gamma secretase inhibitors; nonsteroidal anti-inflammatory agents; anti-inflammatory agents that can reduce neuroinflammation; anti-amyloid antibodies (such as erythromycin, ... such as bapineuzemab, Wyeth/Elan); vitamin E; nicotinic acetylcholine receptor agonists; CB1 receptor inverse agonists or CB1 receptor antagonists; antibiotics; growth hormone secretagogues; histamine H<3> antagonists; AMPA agonists; PDE4 inhibitors; GABA<a> inverse agonists; amyloid aggregation inhibitors; glycogen synthase kinase beta inhibitors; alpha secretase activity promoters; PDE-10 inhibitors; tau kinase inhibitors (e.g., GSK3beta inhibitors, cdk5 inhibitors, or ERK inhibitors); Tau aggregation inhibitors (e.g., REMBER®; RAGE inhibitors (e.g., TTP 488 (PF-4494700)); anti-Ap vaccine; APP ligands; agents that upregulate insulin; cholesterol-lowering agents such as HMG-CoA reductase inhibitors (e.g., statins such as Atorvastatin, Fluvastatin, Lovastatin, Mevastatin, Pitavastatin, Pravastatin, Rosuvastatin, Simvastatin) and/or cholesterol absorption inhibitors (such as Ezetimibe), or combinations of HMG-CoA reductase inhibitors and cholesterol absorption inhibitors (such as, for example, VYTORIN®); fibrates (such as, for example, clofibrate, Clofibride, Etofibrate, and Aluminum Clofibrate); combinations of fibrates and cholesterol-lowering agents and/or cholesterol absorption inhibitors cholesterol; nicotinic receptor agonists; niacin; combinations of niacin and cholesterol absorption inhibitors and/or cholesterol-lowering agents (e.g., SIMCOR® (niacin/simvastatin, available from Abbott Laboratories, Inc.); LXR agonists; <l>RP mimics; H<3> receptor antagonists; histone deacetylase inhibitors; hsp90 inhibitors; 5-HT4 agonists (e.g., PRX-03140 (Epix Pharmaceuticals)); 5-HT6 receptor antagonists; mGluRI receptor modulators or antagonists; mGluR5 receptor modulators or antagonists; mGluR2/3 antagonists; prostaglandin EP2 receptor antagonists; PAI-1 inhibitors; agents that may induce Abeta efflux such as gelsolin; metal protein-buffering compound (e.g., PBT2); and GPR3 modulators; and antihistamines such as Dimebolin (e.g., DIMEBON®, Pfizer).

Accordingly, certain embodiments provide a pharmaceutical composition comprising an effective amount of one or more hydantoins described herein and an additional therapeutic agent, and/or a method of treatment or prophylaxis comprising administering one or more hydantoins described herein together with an additional therapeutic agent wherein the therapeutic agent in the formulation and/or method is disulfiram and/or analogs thereof (see, e.g., USSN 13/213,960 (U.S. Patent Publication No.: US-2012-0071468-A1), and PCT/US2011/048472 (PCT Publication No.: WO 2012/024616)).

Certain embodiments provide a pharmaceutical composition comprising an effective amount of one or more hydantoins described herein and an additional therapeutic agent, and/or a method of treatment or prophylaxis comprising administering one or more hydantoins described herein together with an additional therapeutic agent wherein the therapeutic agent in the formulation and/or method is honokiol and/or analogs thereof (see, e.g., USSN 13/213,960 (U.S. Patent Publication No.: US-2012-0071468-A1), and PCT/US2011/048472 (PCT Publication No.: WO 2012/024616)).

Certain embodiments provide a pharmaceutical composition comprising an effective amount of one or more hydantoins described herein and an additional therapeutic agent, and/or a method of treatment or prophylaxis comprising administering one or more hydantoins described herein together with an additional therapeutic agent wherein the therapeutic agent in the formulation and/or method is tropisetron and/or analogs thereof (see, e.g., USSN 13/213,960 (U.S. Patent Publication No.: US-2012-0071468-A1), and PCT/US2011/048472 (PCT Publication No.: WO 2012/024616)).

Certain embodiments provide a pharmaceutical composition comprising an effective amount of one or more hydantoins described herein and an additional therapeutic agent, and/or a method of treatment or prophylaxis comprising administering one or more hydantoins described herein together with an additional therapeutic agent where the therapeutic agent in the formulation and/or method is tropisetron.

Certain embodiments provide a pharmaceutical composition comprising an effective amount of one or more hydantoins described herein and an additional therapeutic agent, and/or a method of treatment or prophylaxis comprising administering one or more hydantoins described herein together with an additional therapeutic agent wherein the therapeutic agent in the formulation and/or method is nimetazepam and/or analogs thereof (see, e.g., USSN 13/213,960 (U.S. Patent Publication No.: US-2012-0071468-A1), and PCT/US2011/048472 (PCT Publication No.: WO 2012/024616)).

Certain embodiments provide a pharmaceutical composition comprising an effective amount of one or more hydantoins described herein and an additional therapeutic agent, and/or a method of treatment or prophylaxis comprising administering one or more hydantoins described herein together with an additional therapeutic agent where the therapeutic agent in the formulation and/or method is a tropinol ester or related ester (see, e.g., USSN 61/514,381).

Certain embodiments provide a pharmaceutical composition comprising an effective amount of one or more hydantoins described herein and an additional therapeutic agent, and/or a method of treatment or prophylaxis comprising administering one or more hydantoins described herein together with an additional therapeutic agent where the therapeutic agent in the formulation and/or method is a TrkA kinase inhibitor (e.g., ADDN-1351) and/or analogs thereof (see, e.g., USSN 61/525,076).

Certain embodiments provide a pharmaceutical composition comprising an effective amount of one or more hydantoins described herein and an additional therapeutic agent, and/or a method of treatment or prophylaxis comprising administering one or more hydantoins described herein together with an additional therapeutic agent where the therapeutic agent in the formulation and/or method is a D2 receptor agonist and/or an alpha-adrenergic receptor antagonist.

Certain embodiments provide a pharmaceutical composition comprising an effective amount of one or more hydantoins described herein and an additional therapeutic agent, and/or a method of treatment or prophylaxis comprising administering one or more hydantoins described herein together with an additional therapeutic agent where the therapeutic agent in the formulation and/or method is an ASBI as described and/or claimed in USSN 61/728,688, filed Tuesday, November 20, 2012.

Certain embodiments provide a pharmaceutical composition comprising an effective amount of one or more hydantoins described herein and an additional therapeutic agent, and/or a method of treatment or prophylaxis comprising administering one or more hydantoins described herein together with an additional therapeutic agent where the therapeutic agent in the formulation and/or method is one or more cholinesterase inhibitors (e.g., acetyl- and/or butyrylcholinesterase inhibitors).

Certain embodiments provide a pharmaceutical composition comprising an effective amount of one or more hydantoins described herein and an additional therapeutic agent, and/or a method of treatment or prophylaxis comprising administering one or more hydantoins described herein together with an additional therapeutic agent where the therapeutic agent in the formulation and/or method is one or more muscarinic antagonists (e.g., m<1> agonists or m<2> antagonists).

Certain embodiments provide a pharmaceutical composition comprising an effective amount of one or more hydantoins described herein and an additional therapeutic agent, and/or a method of treatment or prophylaxis comprising administering one or more hydantoins described herein together with an additional therapeutic agent wherein the therapeutic agent in the formulation and/or method is one or more compounds selected from the group consisting of cholinesterase inhibitors (such as, for example, (.+-.)-2,3-dihydro-5,6-dimethoxy-2-[[1-(phenylmethyl)-4-piperidinyl]methyl-1]-1H-inden-1-one hydrochloride, i.e., donepezil hydrochloride, available as the ARICEPT® brand of donepezil hydrochloride), N-methyl-D-aspartate receptor inhibitors (such as, for example, Namenda® (memantine HCl)); anti-amyloid antibodies (such as bapineuzumab, gamma-secretase inhibitors, gamma secretase modulators, and beta secretase inhibitors other than hydantoins described herein.

Additional indications.

Use of hydantoin in age-related macular degeneration and glaucoma.

While in various embodiments, the use of APP-binding BACE inhibitors (ABBIs), for example, the various hydantoins described herein, is contemplated for preventing or delaying the onset of a pre-Alzheimer's cognitive condition and/or dysfunction, and/or ameliorating one or more symptoms of a pre-Alzheimer's cognitive condition and/or dysfunction, or preventing or delaying the progression of a pre-Alzheimer's cognitive condition or dysfunction to Alzheimer's disease, and/or for the treatment of Alzheimer's disease, other uses of the ABBIs are also contemplated. In particular, in certain embodiments, the use of the ABBIs is contemplated for the treatment and/or prophylaxis of age-related macular degeneration and/or glaucoma.

Without wishing to be bound by any particular theory, it is thought that abnormal extracellular protein deposition may contribute to the pathogenesis and progression of age-related macular degeneration (AMD), which is also the case in Alzheimer's disease and atherosclerosis. In both conditions, protein deposits contain many shared constituents, such as apoE, complement, and Ap peptides. For example, in human AMD, deposition of the Ap peptide is associated with drusen, where it accumulates and co-localizes with activated complement components (Anderson et al. (2004) Exp. Eye. Res.,78:243-256; Dentchevet al. (2003) Mol. Vis.,9: 184-190; Johnson et al. (2002) Proc Natl Acad Sci USA99: 11830-11835). Luiblet al. (2006) J. Clin. Invest.,116:378-385, showed the presence of potentially toxic amyloid oligomers in drusen, sub-RPE basal deposits, and RPE of human donor eyes using an antibody that specifically recognizes the oligomeric form of Ap. These Ap oligomers were not detected in eyes of age-matched, drusen-free control donors. Isaset al. (2010) Invest. Ophthalmol Vis. Sci.,51:1304-1310, also detected soluble and mature Ap fibrils in drusen. Taken together, these findings implicate Ap in the pathogenesis of AMD. Furthermore, Ap peptide has been detected in sub-RPE basal deposits and neovascular lesions in a mouse model of AMD (Dinget al.(2008)Vision Res.,48:339-345; Maleket al.(2005)Proc Natl Acad Sci USA,102:11900-11905). In this model, aged human APOE4-targeted replacement mice (APOE4 mice) fed a high-fat, cholesterol-enriched (HFC) diet (APOE4-HFC mice) exhibit morphological features observed in both dry and wet AMD. These features include thick, diffuse sub-RPE deposits, focal drusen-like deposits containing lipids and proteins, thickening of Bruch's membrane, irregular regions of RPE atrophy opposite areas of photoreceptor degeneration and CNV (Maleket al.(2005)Proc Natl Acad Sci USA,102: 11900-11905). In the APOE4-HFC mouse AMD model, Ap accumulation is thought to cause damage at the RPE/choroid level, and it has previously been shown that systemic administration of specific anti-Ap40 antibodies can partially attenuate the decline in visual function exhibited in this model (Dinget al.(2008)Vision Res.,48: 339-345). Anti-Ap immunotherapy targeting Ap40 and Ap42 simultaneously has also been shown to block histopathological changes and completely protect visual function in APOE4-HFC mice (Dinget al.(2011) Proc.Nat'l. Acad. Sci. U.S.A.,108(28): E279-E287).

Without wishing to be bound by any particular theory, it is thought that the processing of APP to Ap in the eye occurs through the activities of BACE and Y-secretase in the retina and retinal pigmented epithelial (RPE) cell layers and that sAPPa and Ap are secreted into the vitreous humor (see,

for example, (Prakasamet al.(2008)J. Alzh. Dis.,20: 1243-1253). Ap is further transported into the aqueous humor, where it is easily measured.

In view of these findings, it is believed that ABBIs, for example, the hydantoins described herein, may find use in the treatment or prophylaxis of age-related macular degeneration (AMD) and/or glaucoma. Accordingly, it is believed that ABBIs can be administered to a subject to slow or prevent the onset of AMD (and/or glaucoma), and/or to reduce one or more symptoms of AMD, and/or to slow, stop, or reverse the progression of the disease. In various embodiments, one or more ABBIs (e.g., one or more of the one or more active agents described herein) are administered to a subject (e.g., a human, a non-human mammal) for these purposes. As described above, in various embodiments, the ABBI is administered by a route selected from the group consisting of oral delivery, isophoretic delivery, transdermal delivery, parenteral delivery, aerosol administration, administration via inhalation, intravenous administration, and rectal administration.

In certain embodiments, administration is directly to the eye. Thus, for example, in certain embodiments, the one or more agents may be administered to the eye in the form of eye drops, by intraocular injection, and the like.

Typically, ABBIs are administered in an amount effective for the treatment and/or prophylaxis of AMD or glaucoma, where the effective amount will vary depending on the modality of administration. In certain embodiments, the effective amount is an amount sufficient to mitigate in a mammal one or more symptoms associated with age-related macular degeneration (AMD). In certain embodiments, the effective amount is an amount sufficient to reduce the risk or delay the onset and/or reduce the ultimate severity of an AMD (or glaucoma) disease characterized by reduced Ap in the vitreous and/or aqueous humor and/or amyloid deposits in the retina and/or RPE cell layer.

Test systems for evaluating APP processing

Without wishing to be bound by any particular theory, it is believed that the one or more active agents described herein (e.g., ABBIs, such as the hydantoins described herein) promote the processing of APP by the non-amyloidogenic pathway and/or reduce or inhibit the processing of <a>P<p>by the amyloidogenic pathway. In the non-amyloidogenic pathway, APP is first cleaved by α-secretase within the Ap sequence, releasing the APPsa ectodomain ("sAPPa"). In contrast, the amyloidogenic pathway is initiated when β-secretase cleaves APP at the amino terminus of Ap, thereby releasing the APPsp ectodomain ("sAPPp"). The processing of APP by non-amyloidogenic and amyloidogenic pathways is known in the art and is reviewed, for example, by Xu (2009) J Alzheimers Dis.,16(2): 211-224, and De Strooper,et al.(2010 Nat Rev Neurol6(2): 99-107.

One method of assessing the efficacy of the one or more active agents is to determine a reduction or elimination in the level of APP processing by the amyloidogenic pathway, for example, a reduction or elimination in the level of APP processing by p-secretase cleavage in response to administration of the one or more agents of interest. Assays to determine the extent of APP cleavage at the p-secretase cleavage site are well known in the art. Illustrative assays are described, for example, in U.S. Pat. Nos. 5,744,346 and 5,942,400. Kits for determining the presence and levels in a biological sample of sAPPa and sAPPp, as well as APPneo and Ap are commercially available, for example, from PerkinElmer.

ABBI trial.

The APP-binding BACE inhibitor (ABBI) activity of any of the compounds described herein can be readily verified using, for example, the assays described herein. Basically, in certain embodiments, a pair of assays are used to identify ABBI compounds that inhibit BACE cleavage of the APP substrate MBP-C125, resulting in inhibition of C99 production and the p-site peptide substrate (P5-P5') and also interact with APP, for example, as measured by surface plasmon resonance (SPR) analysis.

In an illustrative embodiment, an APP695wt MBP-C125 fusion protein can be used as one of the substrates and the second substrate can be the commercially available P5-P5' fluorescence substrate. Each of these substrates is incubated with recombinant BACE (R&D (cat. no. 931-AS-050) in, for example, a 96-well plate format. For the MBP-C125 substrate, the C-99 product of BACE cleavage can be measured using an AlphaLisa assay as a readout. For the P5-5' substrate, the loss of fluorescence upon BACE cleavage can be used as a readout. For the SPR assay, the binding analysis of hydantoins to recombinantly prepared APP ectodomain (eAPP) fragments would be performed (Libeuet al.(2012)PLoS ONE7(6):e40027). An ABBI would inhibit BACE cleavage from MBP-C125 and/or the fluorescence substrate and would also bind to the APP ectodomain, such as the APP<230-624> fragment.

Other acellular assays

Illustrative assays that can be used to demonstrate the inhibitory activity of the one or more active agents are described, for example, in WO 2000/017369, WO 2000/0003819 and U.S. Pat. Nos. 5,942,400 and 5,744,346. Such assays can be performed in cell-free incubations or in cellular incubations using cells expressing an alpha-secretase and/or beta-secretase and an APP substrate having alpha-secretase and beta-secretase cleavage sites.

In an illustrative embodiment, the one or more agents of interest are contacted with an APP substrate containing APP alpha-secretase and beta-secretase cleavage sites, e.g., a full-length or variant APP, an APP fragment, or a recombinant or synthetic APP substrate containing the amino acid sequence: KM-DA or NL-DA (APP-SW), incubated in the presence of an alpha-secretase and/or beta-secretase enzyme, a fragment thereof, or a synthetic or recombinant polypeptide variant having alpha-secretase or beta-secretase activity and effective to cleave the alpha-secretase or beta-secretase cleavage sites of APP, under incubation conditions suitable for the cleavage activity of the enzyme. The one or more agents having the desired activity reduce or prevent cleavage of the APP substrate. Suitable substrates optionally include derivatives which may be fusion proteins or peptides containing the substrate peptide and a modification useful to facilitate purification or detection of the peptide or its alpha-secretase and/or beta-secretase cleavage products. Useful modifications include insertion of a known antigenic epitope for antibody binding; attachment of a detectable label or moiety; attachment of a binding substrate and the like.

Suitable incubation conditions for an in vitro acellular assay include, for example: about 200 nanomolar to 10 micromolar of substrate, about 10 to 200 µl of comolar enzyme, and about 0.1 nanomolar to 10 micromolar of the one or more agents, in an aqueous solution, at an approximate pH of 4-7, at about 37°C, for a time period of about 10 minutes to 3 hours. These incubation conditions are illustrative only and may be varied as necessary for the particular assay components and/or the desired measurement system. Optimization of the incubation conditions for the particular assay components should take into account the specific alpha-secretase and/or beta-secretase enzyme used and its pH optimum, any additional enzymes and/or labels that may be used in the assay, and the like. This optimization is routine and will not require undue experimentation.

Another illustrative assay utilizes a fusion peptide having a maltose binding protein (MBP) fused to the C-terminal 125 amino acids of APP-SW. The MBP portion is captured to an assay substrate by an anti-MBP capture antibody. Incubation of the captured fusion protein in the presence of alpha-secretase and/or beta-secretase results in cleavage of the substrate at the alpha-secretase and/or beta-secretase cleavage sites, respectively. This system can be used to screen for inhibitory activity of one or more agents of interest. Analysis of cleavage activity can be performed, for example, by immunoassay of cleavage products. One such immunoassay detects a unique epitope exposed at the carboxy terminus of the cleaved fusion protein, for example, using the SW192 antibody. This assay is described, for example, in U.S. Pat. No. 5,942,400.

Cellular assays

Numerous cell-based assays can be used to assess the activity of the one or more agents of interest on relative alpha-secretase activity relative to beta-secretase activity and/or the processing of APP to release amyloidogenic versus non-amyloidogenic Ap oligomers. Contact of an APP substrate with an alpha-secretase and/or beta-secretase enzyme within the cell and in the presence or absence of the one or more agents can be used to demonstrate the alpha-secretase promoting and/or beta-secretase inhibitory activity of the one or more agents. Preferably, the assay in the presence of the one or more agents provides at least about 30%, most preferably at least about 50% inhibition of enzyme activity, as compared to an uninhibited control.

In one embodiment, cells that naturally express alpha-secretase and/or beta-secretase are used. Alternatively, the cells are modified to express a recombinant alpha-secretase and/or beta-secretase enzyme or synthetic variants, as discussed above. The APP substrate can be added to the culture medium and preferentially expressed in the cells. Cells that naturally express APP, variant or mutant forms of APP, or cells transformed to express an APP isoform, mutant or variant APP, recombinant or synthetic APP, APP fragment or synthetic APP peptide or fusion protein containing the alpha-secretase and/or beta-secretase APP cleavage sites can be used, provided that the expressed APP is allowed to come into contact with the enzyme and can be assayed for enzymatic cleavage activity.

Human cell lines that normally process Ap from APP provide a useful means to assay the inhibitory activities of one or more agents. The production and release of Ap and/or other cleavage products into the culture medium can be measured, for example, by immunoassay, such as Western blotting, or enzyme-linked immunoassay (EIA), such as by ELISA.

Cells expressing an APP substrate and an active alpha-secretase and/or beta-secretase can be incubated in the presence of the agents to demonstrate the relative enzymatic activity of the alpha-secretase and/or beta-secretase compared to a control. The relative activity of the alpha-secretase to beta-secretase can be measured by analysis of one or more cleavage products of the APP substrate. For example, inhibition of beta-secretase activity against the APP substrate would be expected to decrease the release of specific beta-secretase-induced APP cleavage products such as Ap (e.g., Ap40 or Ap42), sAPPp, and APPneo. Promotion or enhancement of alpha-secretase activity against the APP substrate would be expected to increase the release of specific alpha-secretase-induced APP cleavage products such as sAPPa and the p3 peptide.

Although both neural and non-neural cells process and release Ap, levels of endogenous beta-secretase activity are low and often difficult to detect by EIA. Therefore, the use of cell types known to have enhanced beta-secretase activity, enhanced processing of APP to Ap, and/or enhanced Ap production is preferred. For example, transfection of cells with the Swedish mutant form of APP (APP-SW); with the Indiana mutant form (APP-IN); or with APP-SW-IN provides cells that have enhanced beta-secretase activity and produce readily measurable amounts of Ap.

In such assays, for example, cells expressing APP, alpha-secretase and/or beta-secretase are incubated in a culture medium under conditions suitable for the enzymatic activity of alpha-secretase and/or beta-secretase at their cleavage site on the APP substrate. Upon exposure of the cells to the one or more agents, the amount of Ap released into the medium and/or the amount of CTF99 fragments of APP in the cell lysates is reduced compared to the control. APP cleavage products can be analyzed, for example, by immune reactions with specific antibodies, as discussed above.

In certain embodiments, preferred cells for analysis of alpha-secretase and/or beta-secretase activity include primary human neuronal cells, primary transgenic animal neuronal cells where the transgene is APP, and other cells such as those of a stable 293 cell line expressing APP, e.g., APP-SW.

In vivo assays: Animal models

Various animal models can be used to assay the activity of the one or more of interest on the relative activity of alpha-secretase and/or beta-secretase and/or the processing of APP to release Ap. For example, transgenic animals expressing APP substrate, alpha-secretase and/or beta-secretase enzyme can be used to demonstrate the inhibitory activity of the one or more agents. Certain transgenic animal models have been described, for example, in U.S. Pat. Nos. 5,877,399; 5,612,486; 5,387,742; 5,720,936; 5,850,003; 5,877,015. and 5,811,633, and in Ganes et al. (1995) Nature373:523. Animals that exhibit features associated with the pathophysiology of AD are preferred. Administration of the one or more agents to the transgenic mice described herein provides an alternative method for demonstrating inhibitory activity of the one or more agents. Also preferred is administration of the agent(s) in a pharmaceutically effective carrier and by an administrative route that will reach the target tissue in an adequate therapeutic amount.

Inhibition of beta-secretase-mediated APP cleavage at the beta-secretase cleavage site and ApA release can be assayed in these animals by measuring cleavage fragments in the animal's body fluids such as brain fluid or tissues. Similarly, promotion or enhancement of alpha-secretase-mediated APP cleavage at the alpha-secretase cleavage site and sAPPa release can be assayed in these animals by measuring cleavage fragments in the animal's body fluids such as brain fluid or tissues. In certain embodiments, analysis of brain tissues for ApA deposits or plaques is preferred.

By contacting an APP substrate with an alpha-secretase and/or beta-secretase enzyme in the presence of the one or more agents under conditions sufficient to allow enzymatically mediated cleavage of APP and/or release of Ap from the substrate, the one or more desirable agents are effective to reduce beta-secretase-mediated cleavage of APP at the beta-secretase cleavage site and/or are effective to reduce the amounts of Ap released. The one or more agents are also preferably effective to enhance alpha-secretase-mediated cleavage of APP at the alpha-secretase cleavage site and to increase the amounts of sAPPa released. When such contact is administration of the one or more agents to an animal model, for example, as described above, the one or more agents are effective to reduce the deposition of Ap in the brain tissues of the animal, and to reduce the amount and/or size of beta amyloid plaques. When such administration is to a human subject, the one or more agents are effective to inhibit or slow the progression of the disease characterized by increased amounts of Ap, to slow the progression of AD, and/or to prevent the onset or development of AD in a patient at risk for the disease.

Methods for monitoring clinical efficacy

In various embodiments, treatment efficacy can be determined by comparing a baseline measure of a disease parameter prior to administration of the one or more agents (e.g., the hydantoins described herein, or one or more tautomers or stereoisomers thereof, or pharmaceutically acceptable salts or solvates of said one or more hydantoins, said one or more stereoisomers, or said one or more tautomers thereof) and the same parameter beginning one or more time points after the one or more agents or analog has been administered. An exemplary parameter that can be measured is a biomarker (e.g., a peptide oligomer) of APP processing. Such biomarkers include, but are not limited to, increased levels of sAPPa, p3 (Ap17-42 or Ap17-40), sAPPp, soluble Ap40, and/or soluble Ap42 in blood, plasma, serum, urine, mucous membranes, or cerebrospinal fluid (CSF). Detection of increased levels of sAPPa and/or p3, and decreased levels of sAPPp and/or APPneo is an indicator that treatment is effective. Conversely, detection of decreased levels of sAPPa and/or p3, and/or increased levels of sAPPp, APPneo, Tau, or phospho-Tau (pTau) is an indicator that treatment is not effective.

Another parameter for determining treatment efficacy is the level of amyloid plaque deposits in the brain. Amyloid plaques can be determined using any method known in the art, for example, as determined by CT, PET, PIB-PET, and/or MRI. Administration of the one or more agents (e.g., the hydantoins described herein, or one or more tautomers or stereoisomers thereof, or pharmaceutically acceptable salts or solvates of said one or more hydantoins, said one or more stereoisomers, or said one or more tautomers thereof) can result in a reduction in the rate of plaque formation, and even a retraction or reduction of plaque deposits in the brain. Treatment efficacy can also be determined by observing a stabilization and/or improvement of the subject's cognitive abilities. Cognitive abilities may be assessed using any method accepted in the art, including, for example, the Clinical Dementia Rating Scale (CDR), the Mini-Mental State Examination (MMSE), or the Folstein test, assessment criteria listed in the DSM-IV (Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition) or DSM-V, and the like.

Clinical efficacy can be monitored using any method known in the art. Measurable biomarkers to monitor efficacy include, but are not limited to, monitoring blood, plasma, serum, urine, mucosa, or cerebrospinal fluid (CSF) levels of sAPPa, sAPPp, Ap42, Ap40, APPneo, and p3 (e.g., Ap17-42 or Ap17-40). Detection of increased levels of sAPPa and/or p3, and decreased levels of sAPPp and/or APPneo are indicators that the treatment or prevention regimen is effective. Conversely, detection of decreased levels of sAPPa and/or p3, and increased levels of sAPPp and/or APPneo are indicators that the treatment or prevention regimen is not effective. Other biomarkers include Tau and phospho-Tau (pTau). Detection of reduced levels of Tau and pTau are indicators that the treatment or prevention regimen is effective.

Efficacy can also be determined by measuring the amyloid plaque load in the brain. The treatment or prevention regimen is considered effective when the amyloid plaque load in the brain does not increase or decrease. Conversely, the treatment or prevention regimen is considered ineffective when the amyloid plaque load in the brain increases. Amyloid plaque load can be determined using any method known in the art, for example, including CT, PET, PIB-PET, and/or N MRI.

Efficacy may also be determined by measuring the subject's cognitive abilities. Cognitive abilities may be measured using any method known in the art. Illustrative tests include assigning a Clinical Dementia Rating (CDR) score or administering the Mini-Mental State Examination (MMSE) (Folstein, et al., Journal of Psychiatric Research12(3):189-98). Subjects who maintain the same score or who achieve an improved score, for example, by administering the CDR or MMSE, indicate that the treatment or prevention regimen is effective. Conversely, subjects who receive a score indicating decreased cognitive abilities, for example, by administering the CDR or MMSE, indicate that the treatment or prevention regimen has not been effective.

In certain embodiments, the monitoring methods may involve determining a baseline value of a biomarker or measurable parameter (e.g., amyloid plaque load or cognitive abilities) in a subject prior to administering a dose of the one or more agents, and comparing it to a value for the same biomarker or measurable parameter after treatment.

In other methods, a control value (e.g., a mean and standard deviation) of the biomarker or measurable parameter is determined for a control population. In certain embodiments, individuals in the control population are treatment-naive and do not have AD, MCI, or are at risk for developing AD or MCI. In such cases, if the value of the biomarker or measurable clinical parameter approaches the control value, then the treatment is considered effective. In other embodiments, individuals in the control population are treatment-naive and have been diagnosed with AD or MCI. In such cases, if the value of the biomarker or measurable clinical parameter approaches the control value, then the treatment is considered ineffective.

In other methods, a subject who is not currently receiving treatment but who has undergone a prior course of treatment is monitored for one or more of the biomarkers or clinical parameters to determine whether resumption of treatment is required. The measured value of one or more of the biomarkers or clinical parameters in the subject may be compared to a value previously achieved in the subject after a prior course of treatment. Alternatively, the measured value in the subject may be compared to a control value (mean plus standard deviation/ANOVA) determined in a population of subjects after undergoing a course of treatment. Alternatively, the measured value in the subject may be compared to a control value in populations of prophylactically treated subjects who remain free of disease symptoms, or populations of therapeutically treated subjects who show improvement in disease characteristics. In such cases, if the value of the measurable biomarker or clinical parameter approaches the control value, then treatment is considered effective and does not need to be resumed. In all these cases, a significant difference from the control level (e.g., more than one standard deviation) is an indicator that treatment should be resumed in the subject.

In certain embodiments, the tissue sample for analysis is typically blood, plasma, serum, urine, mucous, or cerebrospinal fluid from the subject.

Kits.

In various embodiments, the one or more active agents (e.g., hydantoins) described herein may be included in single or multiple dose containers. The one or more included agents may be provided in kits, for example, including components that may be assembled for use. For example, an active agent may be provided in lyophilized form and a suitable diluent as separate components for combination prior to use. A kit may include an active agent and a second therapeutic agent for co-administration. The active agent and the second therapeutic agent may be provided as separate components. A kit may include a plurality of containers, each container containing one or more unit doses of the compounds. The containers are preferably adapted to the desired mode of administration, including, but not limited to, tablets, gel capsules, sustained release capsules, and the like for oral administration; depot products, prefilled syringes, ampoules, vials, and the like for parenteral administration; and patches, medicated pads, creams and the like for topical administration, for example, as described herein.

In certain embodiments, a kit is provided wherein the kit comprises one or more hydantoin compounds described herein, or a tautomer or stereoisomer thereof, or a pharmaceutically acceptable salt or solvate of said compound, said stereoisomer or said tautomer, preferably provided as a pharmaceutical composition and in a suitable container(s) and/or with a suitable packaging; optionally one or more additional active agents, which, if present, are preferably provided as a pharmaceutical composition and in a suitable container(s) and/or with a suitable packaging; and optionally instructions for use, e.g., written instructions on how to administer the compound(s).

In another embodiment, a kit is provided comprising a single container or multiple containers: (a) a pharmaceutically acceptable composition comprising one or more compounds of claim 1 and/or any of compounds 1-10 shown in Figures 1 and 2, or a tautomer or stereoisomer thereof, or pharmaceutically acceptable salt or solvate of said compound, said stereoisomer or said tautomer, optionally a pharmaceutically acceptable composition comprising one or more additional therapeutic agents; and optionally instructions for use. The kit may optionally comprise labeling (e.g., instructional materials) appropriate for the intended use(s).

As with any pharmaceutical product, the one or more packaging materials and/or the one or more containers are designed to protect the stability of the product during storage and shipping. In addition, the kits may include instructions for use or other informational material that can advise the user such as, for example, a physician, technician, or patient, regarding how to properly administer the one or more compositions as a prophylactic, therapeutic, or ameliorative treatment of the disease of interest. In some embodiments, the instructions may indicate or suggest a dosing regimen including, but not limited to, actual doses and monitoring procedures.

In some embodiments, the instructions may include informational material indicating that administration of the compositions may cause adverse reactions including, but not limited to, allergic reactions such as, for example, anaphylaxis. The informational material may indicate that allergic reactions may manifest only as mild itchy rashes or may be severe and include erythroderma, vasculitis, anaphylaxis, Stevens-Johnson syndrome, and the like. In certain embodiments, the one or more informational material may indicate that anaphylaxis may be fatal and may occur when any foreign protein is introduced into the body. In certain embodiments, the informational material may indicate that these allergic reactions may manifest as hives or rash and develop into fatal systemic reactions and may occur shortly after exposure, such as, for example, within 10 minutes. The literature may further indicate that an allergic reaction may cause a subject to experience paresthesia, hypotension, laryngeal edema, mental status changes, facial or pharyngeal angioedema, airway obstruction, bronchospasm, urticaria and pruritus, serum sickness, arthritis, allergic nephritis, glomerulonephritis, temporal arthritis, eosinophilia, or a combination thereof.

Although instructional materials typically comprise written or printed materials, they are not limited thereto. Contemplated herein are any medium capable of storing such instructions and communicating them to an end user. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD ROMs), and the like. Such media may include addresses of Internet sites that provide such instructional materials.

In some embodiments, the kits may comprise one or more packaging materials such as, for example, a box, bottle, tube, vial, container, spray, insufflator, intravenous (I.V.) bag, sachet, and the like; and at least one unit dosage form of an agent comprising one or more active agents described herein and a packaging material. In some embodiments, the kits also include instructions for using the composition as a prophylactic, therapeutic, or ameliorative treatment for the disease in question.

In some embodiments, the articles of manufacture may comprise one or more packaging materials such as, for example, a box, bottle, tube, vial, container, sprayer, insufflator, intravenous (I.V.) bag, sachet, and the like; and a first composition comprising at least one unit dosage form of an agent comprising one or more hydantoins described herein, or one or more tautomers or stereoisomers thereof, or pharmaceutically acceptable salts or solvates of said one or more hydantoins, said one or more stereoisomers, or said one or more tautomers thereof within the packaging material, together with a second composition comprising a second agent such as, for example, an agent used in the treatment and/or prophylaxis of Alzheimer's disease (e.g., as described herein), or metabolites, analogs, homologs, congeners, derivatives, salts, and combinations thereof. In some embodiments, the articles of manufacture may also include instructions for using the composition as a prophylactic, therapeutic, or ameliorative treatment for the disease in question.

Examples

The following examples are offered to illustrate, but not to limit, the claimed invention.

Example 1

Synthesis of Compound 15-(3.5-difluorophen¡l)-5-phen¡l¡m¡dazol¡din-2.4-d¡one (Hydantoin 1)

Stage 1: Synthesis of (3.5-difluorophen¡l)(2-phen¡l-1.3-dithane-2-¡l)methanol

2-Phenyl-1,3-dithiane (1.791 g, 9.12 mmol) was dissolved in 20 mL of dry THF and cooled to 0 °C. BuLi (6.84 mL, 10.94 mmol) was added dropwise under nitrogen and the mixture was stirred for 30 min at 0 °C. A solution of 3,5-difluorobenzaldehyde (1.00 mL, 9.12 mmol) in THF (10 mL) was added and the mixture was stirred for 30 min, then warmed to room temperature over 1 h and quenched with saturated ammonium chloride solution. The organic phase was washed with brine and dried over sodium sulfate. The solvent was removed in vacuo to give crude (3,5-difluorophenyl)(2-phenyl-1,3-dithian-2-yl)methanol (3.15 g, 9.31 mmol, 104% yield) as a thick yellow oil. The residue was carried on to the next step.

- . - - - - -

(3,5-Difluorophenyl)(2-phenyl-1,3-dithian-2-yl)methanol (3.15 g, 9.31 mmol) was dissolved in 15 mL of acetonitrile and 2.5 mL of water. Bis(trifluoroacetoxy)iodobenzene (5.00 g, 11.63 mmol) in 10 mL of acetonitrile was slowly added at room temperature to the vigorously stirred solution. After 30 min, TLC analysis (25% EtOAc/hexane) indicated complete reaction. EtOAc (150 mL) was added, and the mixture was rinsed with saturated sodium bicarbonate solution (50 mL) and brine (50 mL). The organic fractions were dried, and the solvent was removed in vacuo. The crude product was purified by flash column chromatography (12.5% EtOAc/hexane) to give 2-(3,5-difluorophenyl)-2-hydroxy-1-phenylethanone (1.10 g, 4.43 mmol, 48%) as a pale yellow solid. Proton NMR was consistent with the proposed structure.

Step 3: Synthesis of 1-(3.5-difluorophenyl)-2-phenethylethane-1,2-d¡one

2-(3,5-Difluorophenyl)-2-hydroxy-1-phenylethanone (1.10 g, 4.43 mmol) was dissolved in 80% acetic acid together with diacetoxycopper hydrate (44 mg, 0.22 mmol) and ammonium nitrate (0.30 g, 3.75 mmol). The mixture was heated at reflux for 2.5 hours and then cooled. The reaction mixture was poured into ethyl acetate (50 mL) and washed with brine (2 x 25 mL), dried over sodium sulfate, filtered and evaporated. The crude material was purified by column chromatography (5% EtOAc/hexane) to give 1-(3,5-difluorophenyl)-2-phenylethane-1,2-dione (1.10 g, 4.43 mmol, quant.) as a bright yellow solid. Proton NMR was consistent with the proposed structure.

Step 4: Synthesis of 5-(3,5-difluorophen¡n-5-phenylm¡dazol¡d¡n-2,4-d¡one

To a solution of 1-(3,5-difluorophenyl)-2-phenylethane-1,2-dione (0.99 g, 4.02 mmol), urea (0.435 g, 7.24 mmol) in ethanol (20 mL), and water (5 mL) was added solid NaOH (0.29 g, 7.24 mmol). The reaction mixture was heated to reflux until TLC analysis (50% EtOAc/hexane) indicated complete reaction. The reaction mixture was diluted with water (30 mL) and carefully acidified with 2 M HCl to pH 5. The reaction mixture was extracted with ethyl acetate (100 mL) and washed with water (50 mL) and brine (50 mL). The organic extract was dried over sodium sulfate, filtered and evaporated to give a residue which was triturated with acetone/hexane mixtures to afford 5-(3,5-difluorophenyl)-5-phenylimidazolidine-2,4-dione (0.220 g) as a solid which was highly hydrated with water as determined by NMR spectroscopy. The solid, after heating (120 °C) in vacuo overnight, afforded the desired product (0.20 g, 0.69 mmol, 17%) as a white powder. 1H NMR (400 MHz, d6-DMSO) 8 ppm 11.31 (bs, 1H), 9.44 (s, 1H), 7.37 (m, 6H), 7.10 (d,J = 6.77 Hz, 2H); 13C NMR (100 MHz, d6-DMSO) 8 ppm 173.89, 163.52, 163.39, 161.06, 160.93, 155.78, 143.79, 143.70, 143.61, 139.16, 128.82, 128.44, 126.26, 110.12, 110.04, 109.93, 109.85, 104.11, 103.86, 103.60, 69.43 (note: C-F coupling was observed in several cases, giving rise to doublet and triplet signals; LC (220 nm): Rt = 4.09 min, LC purity: 95.8%, m/z (M-1): 300.3.

Example 2

Synthesis of FAH-2: 2-amino-4-(4-(difluoromethox¡)phen¡l)-4-(3.5-difluorophen¡l)-1-methyl-1H-¡m dazole-5(4H)-one

Step 1: Synthesis of 2-(3.5-difluorophenyl)-1,3-dithane

BF3.OMe2 (0.70 mL, 7.62 mmol) was added dropwise to a solution of 1,3-propanedithiol (0.90 mL, 8.94 mmol) and 3,5-difluorobenzaldehyde (1.00 mL, 8.94 mmol) in DCM (50 mL) at 0 °C. The reaction was stirred at room temperature for 1 h, where TLC analysis (5% EtOAc/hexane) indicated complete reaction. The reaction mixture was then diluted with DCM (50 mL), filtered through Celite (and the Celite pad was washed with additional DCM (3 x 20 mL) and the filtrate was washed with brine (50 mL), saturated NaHCO3 (3 x 50 mL), 10% KOH solution (50 mL), water (50 mL), brine (50 mL) and finally dried over sodium sulfate. The organic extract was filtered and evaporated to afford 2-(3,5-difluorophenyl)-1,3-dithiane (2.14 g, 9.21 mmol, 103%) as white needle-like crystalline solids. Proton NMR was consistent with the proposed structure.

Stage 2: Synthesis of (4-(difluoromethoxy¡)phen¡l)(2-(3.5-difluorophen¡l)-1.3-dithane-2-¡l)methanol

2-(3,5-Difluorophenyl)-1,3-dithiane (2.14 g, 9.21 mmol) was dissolved in 20 mL of dry THF and cooled to 0 °C. BuLi (8.50 mL, 10.20 mmol) was added dropwise under nitrogen and the mixture was stirred for 15 min at 0 °C. A solution of 4-(difluoromethoxy)benzaldehyde (1.30 mL, 9.33 mmol) in THF (10 mL) was added and the mixture was stirred for 10 min, then warmed to room temperature over 10 min and quenched with saturated ammonium chloride solution. The organic phase was washed with brine and dried over sodium sulfate. The solvent was removed in vacuo to give a residue which was purified by flash column chromatography (10% EtOAc/hexane) to provide (4-(difluoromethoxy)phenyl)(2-(3,5-difluorophenyl)-1,3-dithian-2-yl)methanol (1.90 g, 4.70 mmol, 51%) as a thick yellow oil. Proton NMR was consistent with the proposed structure.

Step 3: Synthesis of 2-(4-(difluoromethoxy)phenyl)-1-(3.5-difluorophenoxy)-2-hydroxyethanone

(4-(Difluoromethoxy)phenyl)(2-(3,5-difluorophenyl)-1,3-dithian-2-yl)methanol (1.90 g, 4.70 mmol) was dissolved in 15 mL of acetonitrile and 2.5 mL of water. Bis(trifluoroacetoxy)iodobenzene (2.53 g, 5.87 mmol) in 10 mL of acetonitrile was slowly added at room temperature to the vigorously stirred solution. After 30 min, TLC analysis (25% EtOAc/hexane) indicated complete reaction. EtOAc (150 mL) was added, and the mixture was rinsed with saturated sodium bicarbonate solution (50 mL) and brine (50 mL). The organic fractions were dried, and the solvent was removed in vacuo. The crude product was purified by flash column chromatography (12.5% EtOAc/hexane) to give 2-(4-(difluoromethoxy)phenyl)-1-(3,5-difluorophenyl)-2-hydroxyethanone (0.460 g, 1.46 mmol, 31%) as a pale yellow solid. Proton NMR was consistent with the proposed structure.

Step 4: Synthesis of 1-(4-(difluoromethoxy)phenyl)-2-(3,5-difluorophenyl)ethane-1,2-dione

2-(4-(Difluoromethoxy)phenyl)-1-(3,5-difluorophenyl)-2-hydroxyethanone (0.46 g, 1.46 mmol) was dissolved in 80% acetic acid together with diacetoxycopper hydrate (26 mg, 0.13 mmol) and ammonium nitrate (0.18 g, 2.25 mmol). The mixture was heated at reflux for 90 minutes and then cooled. The reaction mixture was poured into ethyl acetate (50 mL) and washed with brine (2 x 25 mL), dried over sodium sulfate, filtered and evaporated. The crude material was passed through a plug of silica gel, evaporated and azeotroped with toluene (3 x 20 mL) to remove excess acetic acid to give crude 1-(4-(difluoromethoxy)phenyl)-2-(3,5-difluorophenyl)ethane-1,2-dione (0.456 g, 1.46 mmol, 100%) as a bright yellow solid. The crude solid was carried on to the next step.

Stage 5: Synthesis of 2-amino-4-(4-(difluoromethox¡)phen¡l)-4-(3.5-difluorophen¡l)-1-methyl-1H-m¡ dazole-5(4H)-one

To 1-(4-(difluoromethoxy)phenyl)-2-(3,5-difluorophenyl)ethane-1,2-dione (0.456 g, 1.46 mmol) in ethanol (20 mL) and water (5 mL) were added 1-methylguanidine hydrochloride (0.16 g, 1.46 mmol) and potassium carbonate (0.61 g, 4.38 mmol). The mixture was refluxed for 3 hours and then cooled to room temperature. The volatiles were removed in vacuo and the residue was taken up in water and extracted into chloroform (50 mL). The organic fractions were dried over sodium sulfate and the solvent was removed in vacuo. The crude material was purified by column chromatography (EtOAc) to afford 2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3,5-difluorophenyl)-1-methyl-1H-imidazol-5(4H)-one (0.172 g, 0.47 mmol) as a glass which was heavily contaminated with ethyl acetate as determined by NMR analysis. The glass was taken up in dry ethanol (3 mL) and layered with hexane (1 mL) to give a cloudy solution. The solution was rotary evaporated to give an oil which solidified on standing. This was dried overnight and the weight obtained was 0.150 g. The solid contained solvent residues of ethanol and hexane as determined by NMR analysis. Therefore, the solids were redissolved in iso-propanol, rotary evaporated and dried under high vacuum at 90 °C overnight to provide 2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3,5-difluorophenyl)-1-methyl-1H-imidazol-5(4H)-one (0.130 g, 0.35 mmol, 24%) as an off-white solid. 1H NMR (400 MHz, CDCls) 8 ppm 7.47 (d,J =8.79 Hz, 2H), 7.10-7.00 (m, 4H), 6.70 (tt,J= 8.73, 2.32 Hz, 1H), 6.53 (t,Jh-f =73.76 Hz, 1H), 5.45 (s a, 1H), 3.11 (s, 3H); 13C NMR (100 MHz, CDCla) 8 ppm 178,619, 164,122, 163,996, 161,650, 161,524, 155,704, 150,742, 150,714, 150,687, 145,164, 145,081, .991, 137.735, 128.390, 119.444, 118.328, 115.743, 113.158, 110.329, 110.255, 110.138, 110.065, 103.421, 103.169, 102.917, 77.203, 25.966 (note: C-F coupling was observed in several cases, giving rise to doublet and triplet signals); LC (260 nm): Rt = 3.899 min, LC purity: 96.3%, m/z (M-1): 366.

Example 3

Synthesis of FAH-3: 2-amino-4-(4-(difluoromethox¡)phen¡l)-4-(3.5-difluorophen¡l)-1-methyl-1H-¡m ¡dazol-5(4H)-oneStage 1: Synthesis of 2-(3.5-difluorophen¡l)-1.3-dithane

BF3.OMe2 (1.40 mL, 15.25 mmol) was added dropwise to a solution of 1,3-propanedithiol (1.81 mL, 17.87 mmol) and 3,5-difluorobenzaldehyde (2.00 mL, 17.87 mmol) in DCM (50 mL) at 0 °C. The reaction was stirred at room temperature for 1 h, where TLC analysis (5% EtOAc/hexane) indicated complete reaction. The reaction mixture was then diluted with additional DCM (50 mL), filtered through Celite, and the Celite pad was washed with additional DCM (3 x 20 mL). The filtrate was washed with saturated NaHCO3 (3 x 50 mL), 10% KOH solution (2 x 50 mL), water (2 x 50 mL), brine (50 mL) and finally dried over sodium sulfate. The organic extract was filtered and evaporated to afford 2-(3,5-difluorophenyl)-1,3-dithiane (4.12 g, 17.73 mmol, 99%) as a white solid. Proton NMR was consistent with the proposed structure.

Step 2: Synthesis of 4-(difluoromethoxy)-3-methylbenzaldehyde

Attempt 1:

A solution of sodium chlorodifluoroacetate (3.23 g, 21.15 mmol) and 4-hydroxy-3-methylbenzaldehyde (1.44 g, 10.58 mmol), potassium carbonate (2.19 g, 15.87 mmol) in a mixture of DMF (8 mL) and water (2 mL) was heated at 100 °C for 2 h. The reaction mixture was cooled and conc. HCl (1.5 mL) was added followed by water (2.1 mL). The reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (3 x 25 mL). The organic extract was washed with 10% (m/v) aqueous LiCl solution (3 x 25 mL), dried over sodium sulfate, filtered, and evaporated to give a residue which was subjected to flash chromatography (15% EtOAc/hexane) to give 4-(difluoromethoxy)-3-methylbenzaldehyde (0.244 g, 1.31 mmol, 12%) as a brown oil and 4-hydroxy-3-methylbenzaldehyde (1.164 g, 8.55 mmol, 81%) was recovered as a brown solid.

The experiment was repeated again, except for the absence of water. Briefly, the experimental procedure is given below:

Attempt 2:

A solution of sodium chlorodifluoroacetate (2.60 g, 17.04 mmol) and 4-hydroxy-3-methylbenzaldehyde (1.16 g, 8.52 mmol) in DMF (15 mL) was added over 3 hours to a solution of DMF (15 mL) containing potassium carbonate (1.77 g, 12.78 mmol) at 95 °C. The reaction was allowed to proceed for a further 15 minutes and then cooled. The reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (3 x 50 mL). The organic extract was washed with 10% (m/v) aqueous LiCl solution (3 x 25 mL), dried over sodium sulfate, filtered, and evaporated to give a residue which was subjected to flash chromatography (15% EtOAc/hexane) to give 4-(difluoromethoxy)-3-methylbenzaldehyde (021GLM-053_1(2), 1.00 g, 5.37 mmol, 63%) as a yellow oil. This oil was combined with that from the previous experiment and passed through a Pasteur pipette column eluting with 10% EtOAc/hexane to give an oil that solidified upon standing (1.315 g, 7.06 mmol, 67% for the two reactions). Proton NMR was consistent with the proposed structure.

Finally, repeating the experiment using the conditions of attempt 2, an additional 1.4 g of the desired product was isolated.

Stage 3: Synthesis of ^-(difluoromethoxh^-methylpheninté-pho^-difluorophenin-l^-dithian^-immethanol

2-(3,5-DifluorophenM)-1,3-dithiane (3.12 g, 13.43 mmol) was dissolved in 30 mL of dry THF and cooled to -10 °C. BuLi (1.6 M, 12.0 mL, 19.20 mmol) was added dropwise under nitrogen and the mixture was stirred for 15 min at -10 °C to give a blood-red solution. A solution of 4-(difluoromethoxy)-3-methylbenzaldehyde (2.50 g, 13.43 mmol) in THF (10 mL) was added dropwise and the mixture was stirred for 15 min, then warmed to room temperature for 10 min and quenched with saturated ammonium chloride solution. The organic phase was washed with brine and dried over sodium sulfate. The solvent was removed and the residue was subjected to flash chromatography (10% EtOAc/hexane) to give (4-(difluoromethoxy)-3-methylphenyl)(2-(3,5-difluorophenyl)-1,3-dithian-2-yl)methanol (3.07 g, 7.22 mmol, 55%) as a thick oil that solidified upon standing. NMR was consistent with the proposed structure.

Step 4: Synthesis of 2-(4-(difluoromethoxy¡)-3-methylphen¡l)-1-(3.5-difluorophen¡l)-2-hydroxy¡ethanone

(4-(Difluoromethoxy)-3-methylphenyl)(2-(3,5-difluorophenyl)-1,3-dithian-2-yl)methanol (3.07 g, 7.34 mmol) was dissolved in acetonitrile (15 mL) and water (2.5 mL). Bis(trifluoroacetoxy)iodobenzene (3.94 g, 9.17 mmol) in acetonitrile (10 mL) was slowly added at room temperature to the vigorously stirred solution. After 20 min, TLC analysis (20% EtOAc/hexane) indicated complete reaction. EtOAc (150 mL) was added, and the mixture was rinsed with saturated sodium bicarbonate solution (50 mL) and brine (50 mL). The organic fractions were dried, and the solvent was removed in vacuo. The crude product was purified twice by flash column chromatography (10% EtOAc/hexane) to give 2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3,5-difluorophenyl)-2-hydroxyethanone (0.853 g, 2.60 mmol, 35%) as a pale yellow oil. Proton NMR was consistent with the proposed structure.

Step 5: Synthesis of 1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3.5-difluorophenyl)ethane-1,2-d¡one

2-(4-(Difluoromethoxy)-3-methylphenyl)-1-(3,5-difluorophenyl)-2-hydroxyethanone (0.853 g, 2.60 mmol) was dissolved in 80% acetic acid together with diacetoxycopper hydrate (52 mg, 0.26 mmol) and ammonium nitrate (0.156 g, 1.95 mmol). The mixture was heated at reflux for 90 minutes and then cooled. The reaction mixture was poured into ethyl acetate (50 mL) and washed with brine (2 x 25 mL), dried over sodium sulfate, filtered and evaporated. The residue was azeotroped with toluene to remove acetic acid and the residue (0.737 g, 2.26 mmol, 87%) was used directly in the next step.

Step 6: Synthesis of 2-amino-4-(4-(difluoromethox)-3-methylphenyl)-4-(3.5-difluorophenyl)-1-methyl 1H-¡m¡dazol-5(4H)-one

To a mixture of 1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3,5-difluorophenyl)ethane-1,2-dione (737 mg, 2.26 mmol) in ethanol (15 mL) and dioxane (15 mL) was added 1-methylguanidine hydrochloride (990 mg, 9.04 mmol) and stirred at room temperature for 15 minutes. Sodium carbonate (958 mg, 9.04 mmol) in water (5 mL) was added and the mixture was immersed in an oil bath at 85 °C and stirred for 3 hours. TLC analysis (EtOAc) indicated complete reaction. The reaction mixture was cooled to room temperature and concentrated. Purification by flash chromatography (EtOAc) afforded 2-amino-4-(4-(difluoromethoxy)-3-methylphenyl)-4-(3,5-difluorophenyl)-1-methyl-1H-imidazol-5(4H)-one (0.52 g, 1.36 mmol, 60%) as a yellow solid. 1H NMR (400 MHz, CDCb) 5 ppm 7.34, 7.24 (m, 2H), 7.08-6.97 (m, 3H), 6.74-6.66 (m, 1H), 6.47 (t,Jh-f =74.03 Hz, 1H), 5.45 (bs, 2H), 3.11 (s, 3H), 2.25 (s, 3H); Le (260 nm): Tr = 3.919 min, LC purity: 96.1%, m/z (M+1): 382, LC (220 nm): Tr = 3.922 min, LC purity: 96.8%.

Example 4

Synthesis of FAH-5: 2-amino-4-(3.5-difluorophenyl)-4-(3.5-dimethylphenyl)-1-methyl-1H-imidazole-5(4H)-one Synthesis of 2-(3.5-difluorophenyl)- 1.3-dithyan

BF3.OMe2 (2.50 mL, 27.5 mmol) was added dropwise to a solution of 1,3-propanedithiol (3.70 mL, 36.6 mmol) and 3,5-difluorobenzaldehyde (4.10 mL, 36.6 mmol) in DCM (75 mL) at 0 °C. The reaction was stirred at room temperature for 1 h, where TLC analysis (5% EtOAc/hexane) indicated complete reaction. The reaction mixture was then diluted with additional DCM (50 mL), filtered through Celite, and the Celite pad was washed with additional DCM (3 x 50 mL). The filtrate was washed with saturated NaHCO3 (3 x 100 mL), 10% KOH solution (2 x 100 mL), water (100 mL), brine (100 mL) and finally dried over sodium sulfate. The organic extract was filtered through a pad of silica and the silica pad was washed with 10% ethyl acetate/hexane mixtures (3 x 20 mL). The organic extract was evaporated to afford 2-(3,5-difluorophenyl)-1,3-dithiane (8.44 g, 36.3 mmol, 99%) as a white crystalline solid. NMR was consistent with the proposed structure.

Synthesis of (2-(3.5-difluorophenyl)-1.3-dithian-2-ylH3.5-dimethylphenyl)methanol

2-(3,5-Difluorophenyl)-1,3-dithiane (8.00 g, 34.4 mmol) was dissolved in 100 mL of dry THF and cooled to -10 °C. BuLi (1.6 M, 34 mL, 54.4 mmol) was added dropwise under nitrogen and the mixture was stirred for 15 min at -10 °C to give a brown solution. A solution of 3,5-dimethylbenzaldehyde (4.84 g, 36.1 mmol) in THF (10 mL) was added dropwise and the reaction mixture was stirred for 15 min, then warmed to room temperature over 30 min and quenched with saturated ammonium chloride solution. The organic phase was washed with brine and dried over sodium sulfate. The solvent was removed and the residue was purified by flash chromatography (10% EtOAc/hexane) to give (2-(3,5-difluorophenyl)-1,3-dithian-2-yl)(3,5-dimethylphenyl)methanol (6.60 g, 18.01 mmol, 52%) as a thick oil that solidified upon standing. NMR was consistent with the proposed structure.

Synthesis of 1-(3.5-difluorophen¡l)-2-(3.5-dimethylphen¡l)-2-hydroxy¡ethanone

(2-(3,5-Difluorophenyl)-1,3-dithian-2-yl)(3,5-dimethylphenyl)methanol (6.60 g, 18.01 mmol) was dissolved in a solution of acetonitrile (75 mL) and water (15 mL). Bis(trifluoroacetoxy)iodobenzene (9.68 g, 22.51 mmol) was added in several portions to the vigorously stirred solution at room temperature. After 60 minutes, TLC analysis (20% EtOAc/hexane) appeared to indicate complete reaction. Ethyl acetate (150 mL) was added and the mixture was rinsed with saturated sodium bicarbonate solution (2 x 50 mL) and brine (50 mL). The organic fractions were dried over sodium sulfate, filtered, and evaporated. The residue was purified twice by flash column chromatography (10% EtOAc/hexane) to give 1-(3,5-difluorophenyl)-2-(3,5-dimethylphenyl)-2-hydroxyethanone (2.10 g, 7.60 mmol, 42%, ca. 90% purity by NMR) as a pale yellow solid contaminated with starting material (ca. 10%); Fr (10% EtOAc/hexane): 0.20, which was identical for both starting material and product. However, NMR was consistent with the proposed structure of the product, which was the major component.

Synthesis of 1-(3.5-difluorophen¡l)-2-(3.5-dimethylphen¡l)ethane-1.2-dione

1-(3,5-Difluorophenyl)-2-(3,5-dimethylphenyl)-2-hydroxyethanone (2.10 g, 7.60 mmol) was dissolved in 80% acetic acid (10 mL) together with diacetoxycopper hydrate (0.15 g, 0.76 mmol) and ammonium nitrate (0.46 g, 5.70 mmol). The mixture was heated at reflux for 90 minutes and then cooled. The green reaction mixture was poured into ethyl acetate (50 mL), washed with brine (2 x 25 mL), dried over sodium sulfate, filtered and evaporated. The residue was subjected to flash chromatography (20% EtOAc/hexane) to provide 1-(3,5-difluorophenyl)-2-(3,5-dimethylphenyl)ethane-1,2-dione (1.13 g, 4.12 mmol, 54%) as a yellow solid. NMR was consistent with the proposed structure.

Synthesis of 2-amino-4-(3.5-difluorophen¡l)-4-(3.5-dimethylphen¡l)-1-methyl-1H-midazole-5(4H )-one (FAH5)

To a mixture of 1-(3,5-difluorophenyl)-2-(3,5-dimethylphenyl)ethane-1,2-dione (500 mg, 1.82 mmol) in ethanol (15 mL) and dioxane (15 mL) was added 1-methylguanidine hydrochloride (799 mg, 7.29 mmol) and stirred at room temperature for 15 minutes. Sodium carbonate (773 mg, 7.29 mmol) in water (5 mL) was added and the mixture was immersed in an oil bath at 85 °C and stirred for 4 hours. TLC (EtOAc) analysis indicated complete reaction. The reaction mixture was cooled to room temperature and concentrated. The residue was purified twice by column chromatography (EtOAc, 50% EtOAc/hexane) and finally by PTLC (chloroform) to afford 2-amino-4-(3,5-difluorophenyl)-4-(3,5-dimethylphenyl)-1-methyl-1H-imidazol-5(4H)-one (0.20 g, 0.61 mmol, 33%) as a white solid after drying under high vacuum at 60 °C for 36 h. 1H NMR (400 MHz,CDCI3)5 ppm 7.07 (d,J =6.83 Hz, 2H), 7.02 (s a, 2H), 6.91 (s a, 1H), 6.69 (t,J =8.48 Hz, 1H), 5.22 (s, 2H), 3.10 (s, 3H), 2.27 (s, 6H); 13C NMR (100 MHz, CDCl3) 5 ppm 164.1, 163.9, 161.6, 161.5, 155.3, 145.4, 140.4, 138.2, 129.7, 124.5, 110.5, 110.4, 110.3, 110.2, 103.0, 77.2, 25.9, 21.41, (note: due to the presence of fluorine atoms, J<2c-f>-J<4c-f> couplings are seen giving rise to poorly resolved triplets and doublets); LC (230 nm) Tr (min) = 3.97, purity by LC = 95.29%; m/z:[M+H]+ found = 330.1, [M+H]+ expected = 330.1 (C<18>H<18>F<2>N<3>O).

Example 5

Synthesis of FAH-4 (ITH002329)

Synthesis of 2-(3.5-difluorophenyl)-1.3-dithane

BF3.OMe2 (1.40 mL, 15.25 mmol) was added dropwise to a solution of 1,3-propanedithiol (1.81 mL, 17.87 mmol) and 3,5-difluorobenzaldehyde (2.00 mL, 17.87 mmol) in DCM (50 mL) at 0 °C. The reaction was stirred at room temperature for 1 h, where TLC analysis (5% EtOAc/hexane) indicated complete reaction. The reaction mixture was then diluted with additional DCM (50 mL), filtered through Celite, and the Celite pad was washed with additional DCM (3 x 20 mL). The filtrate was washed with saturated NaHCO3 (3 x 50 mL), 10% KOH solution (2 x 50 mL), water (2 x 50 mL), brine (50 mL) and finally dried over sodium sulfate. The organic extract was filtered and evaporated to afford 2-(3,5-difluorophenyl)-1,3-dithiane (4.12 g, 17.73 mmol, 99%) as a white solid.

Synthesis of 3-(difluoromethoxy)benzaldehyde

A solution of sodium chlorodifluoroacetate (12.48 g, 82 mmol) and 3-hydroxybenzaldehyde (5.00 g, 40.9 mmol) in DMF (75 mL) was added over 3 hours to a solution of DMF (25 mL) containing potassium carbonate (8.49 g, 61.4 mmol) at 95 °C. The reaction was allowed to proceed for a further 2 hours and then cooled. The reaction mixture was diluted with water (100 mL) and extracted with ethyl acetate (4 x 50 mL). The organic extract was washed with 10% (m/v) aqueous LiCl solution (3 × 25 mL), dried over sodium sulfate, filtered, and evaporated to give a residue which was subjected to flash chromatography (15% EtOAc/hexane) to give 3-(difluoromethoxy)benzaldehyde (2.50 g, 14.52 mmol, 36%) as a yellow oil. NMR was consistent with the proposed structure.

Synthesis of (3-(difluoromethoxy)phenyl)(2-(3.5-difluorophenyl)-1.3-dithian-2-yl)methanol

2-(3,5-Difluorophenyl)-1,3-dithiane (3.37 g, 14.52 mmol) was dissolved in 30 mL of dry THF and cooled to -10 °C. BuLi (1.6 M, 12.0 mL, 19.20 mmol) was added dropwise under nitrogen and the mixture was stirred for 15 min at -10 °C to give a blood-red solution. A solution of 3-(difluoromethoxy)benzaldehyde (2.50 g, 14.52 mmol) in THF (10 mL) was added dropwise and the mixture was stirred for 15 min, then warmed to room temperature over 10 min and quenched with saturated ammonium chloride solution. The organic phase was washed with brine and dried over sodium sulfate. The solvent was removed and the residue was subjected to flash chromatography (10% EtOAc/hexane) to give (3-(difluoromethoxy)phenyl)(2-(3,5-difluorophenyl)-1,3-dithian-2-yl)methanol (3.33 g, 8.23 mmol, 57%) as a thick oil that solidified upon standing. NMR was consistent with the proposed structure.

Synthesis of 2-(3-(difluoromethoxy)phenyl)-1-(3.5-difluorophenyl)-2-hydroxyethanone

(3-(Difluoromethoxy)phenyl)(2-(3,5-difluorophenyl)-1,3-dithian-2-yl)methanol (3.33 g, 8.23 mmol) was dissolved in 15 mL of acetonitrile and 2.5 mL of water. Bis(trifluoroacetoxy)iodobenzene (4.43 g, 10.29 mmol) in 10 mL of acetonitrile was slowly added to the vigorously stirred solution at room temperature. After 30 min, TLC analysis (20% EtOAc/hexane) indicated complete reaction. EtOAc (150 mL) was added, and the mixture was rinsed with saturated sodium bicarbonate solution (50 mL) and brine (50 mL). The organic fractions were dried, and the solvent was removed in vacuo. The crude product was purified twice by flash column chromatography (10% EtOAc/hexane) to give 2-(3-(difluoromethoxy)phenyl)-1-(3,5-difluorophenyl)-2-hydroxyethanone (1.01 g, 3.21 mmol, 39%) as a pale yellow oil. The r Mn was consistent with the proposed structure.

Synthesis of 1-(3-(difluoromethoxy)phenyl)-2-(3.5-difluorophenyl)-1,2-dione

2-(3-(Difluoromethoxy)phenyl)-1-(3,5-difluorophenyl)-2-hydroxyethanone (1.00 g, 3.18 mmol) was dissolved in 80% acetic acid (10 mL) together with diacetoxycopper hydrate (0.13 g, 0.64 mmol) and ammonium nitrate (0.19 g, 2.39 mmol). The mixture was heated at reflux for 90 minutes and then cooled. The copper-colored reaction mixture was poured into ethyl acetate (50 mL), washed with brine (2 x 25 mL), dried over sodium sulfate, filtered, and evaporated. The residue was subjected to flash chromatography (20% EtOAc/hexane) to provide 1-(3-(difluoromethoxy)phenyl)-2-(3,5-difluorophenyl)ethane-1,2-dione (0.46 g, 1.48 mmol, 47%) as a yellow oil. Further elution from the column provided starting material (0.40 g, 1.27 mmol, 40% recovery) as an oil. The desired product was used as received.

Synthesis of 2-amino-4-(3-(difluoromethoxy)phen-4-(3.5-difluorophenyl)-1-methyl-1H-midazole-5( 4H)-one

To a mixture of 1-(3-(difluoromethoxy)phenyl)-2-(3,5-difluorophenyl)ethane-1,2-dione (463 mg, 1.48 mmol) in ethanol (15 mL) and dioxane (15 mL) was added 1-methylguanidine hydrochloride (650 mg, 5.93 mmol) and stirred at room temperature for 15 minutes. Sodium carbonate (629 mg, 5.93 mmol) in water (5 mL) was added and the mixture was immersed in an oil bath at 85 °C and stirred for 3 hours. TLC (EtOAc) analysis indicated complete reaction. The reaction mixture was cooled to room temperature and concentrated. The residue was purified twice by LCP (EtOAc) to afford 2-amino-4-(3-(difluoromethoxy)phenyl)-4-(3,5-difluorophenyl)-1-methyl-1H-imidazol-5(4H)-one (0.22 g, 0.60 mmol, 40%) as a yellow solid after drying under high vacuum at 60 °C for 48 h.

1H NMR (400 MHz, CDCla) 8 ppm 7.38-7.29 (m, 2H), 7.24 (m wide, 1H), 7.10-7.00 (m, 3H), 6.71 ( m, 1H), 6.49 (t,Jh-f =74.03 Hz, 1H), 5.61 (s a, 2H), 3.10 (s, 3H); 13C NMR (100 MHz, CDCla) 8 ppm 178.30, 164.13, 164.00, 161.66, 161.53, 156.03, 151.27, 151.24, 151.21, 145.00, 144.92, 144.83, 142.88, 129.90, 123.81, 118.73, 118.42, 118.17, 115.84, 113.25, 110.30, 110.22, 110, 11, 110.03, 103.47, 103.22, 102.97, 74.92, 25.95 (note: due to the presence of fluorine atoms, J<2c-f>- J<4c-f> couplings are observed, giving rise to triplets and doublets); LC (220 nm): Tr = 3, 85 min, LC purity: 95.6%, m/z [M]+ = 367.9.

Example 6

FAH-17: 2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3-fluorophenyl)-1-methyl-1H-midazole-5 (4H)-one

Step 1: Synthesis of 2-(4-(difluoromethoxy)-3-methylphenyl)-1,3-dithane

BF3.OEt2 (4.30 mL, 34.8 mmol) was added dropwise to a solution of 1,3-propanedithiol (4.07 mL, 40.3 mmol) and 3-fluorobenzaldehyde (5.00 g, 40.3 mmol) in DCM (201 mL) at 0 °C. The reaction was stirred at room temperature for 1 h, where TLC analysis (5% EtOAc/hexane) indicated complete reaction. The reaction mixture was then diluted with DCM (50 mL), filtered through Celite (and the Celite pad was washed with additional DCM (3 x 50 mL)) and the filtrate was washed with brine (100 mL), saturated NaHCO3 (3 x 100 mL), 10% KOH solution (100 mL), water (100 mL), brine (100 mL) and finally dried over sodium sulfate. The organic extract was filtered and evaporated. The product was purified using 5% ethyl acetate:hexane to afford 2-(3-fluorophenyl)-1,3-dithiane (8.71 g, 39.0 mmol, 97%) as a slightly clear oil. The oil was used directly in the next step. NMR was consistent with the proposed structure.

Step 2: Synthesis of 4-(difluoromethoxy)-3-methylbenzaldehyde

A solution of sodium chlorodifluoroacetate (2.60 g, 17.04 mmol) and 4-hydroxy-3-methylbenzaldehyde (1.16 g, 8.52 mmol) in DMF (15 mL) was added over 3 hours to a solution of DMF (15 mL) containing potassium carbonate (1.77 g, 12.78 mmol) at 95 °C. The reaction was allowed to proceed for a further 15 minutes and then cooled. The reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (3 x 50 mL). The organic extract was washed with 10% (m/v) aqueous LiCl solution (3 x 25 mL), dried over sodium sulfate, filtered, and evaporated to give a residue which was subjected to flash chromatography (15% EtOAc/hexane) to give 4-(difluoromethoxy)-3-methylbenzaldehyde (021GLM-053_1(2), 1.00 g, 5.37 mmol, 63%) as a yellow oil. This oil was combined with that from the previous experiment and passed through a Pasteur pipette column eluting with 10% EtOAc/hexane to give an oil that solidified upon standing (1.315 g, 7.06 mmol, 67% for the two reactions). Proton NMR was consistent with the proposed structure.

Finally, repeating the experiment using the conditions of attempt 2, an additional 1.4 g of the desired product was isolated.

Stage 3: Synthesis of (2-(4-(difluoromethoxy¡)-3-methylphen¡l)-1.3-dithan-2-¡l)(3-fluorophen¡hmethanol

2-(3-Fluorophenyl)-1,3-dithiane (4.00 g, 18.66 mmol) was dissolved in dry THF (93.5 mL) and cooled to -10 °C. nBuLi (1.6 M, 14.00 mL, 22.40 mmol) was added dropwise under a nitrogen atmosphere and the mixture was stirred for 30 min at -10 °C to give a deep red solution. A solution of 4-(Difluoromethoxy)-3-methylbenzaldehyde (3.47 g, 18.66 mmol) in THF (93.5 mL) was added dropwise and the mixture was allowed to cool to -10 °C and stirred for 15 min, then warmed to room temperature over 1 h and quenched with saturated ammonium chloride solution (7.5 mL) followed by dilution with EtOAc (50 mL). The organic phase was washed with water (2 x 20 mL) and brine (1 x 20 mL) and dried over sodium sulfate. After filtration and concentration, the crude product was purified by flash column chromatography (15% EtOAc/Hex) to give (4-(difluoromethoxy)-3-methylphenyl)(2-(3-fluorophenyl)-1,3-dithian-2-yl)methanol (6.00 g, 14.96 mmol, 80%) as an oil.

Step 4: Synthesis of 2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3-fluorophenyl)-2-hydroxyethanone

(4-(Difluoromethoxy)-3-methylphenyl)(2-(3-difluorophenyl)-1,3-dithian-2-yl)methanol (3.07 g, 7.34 mmol) was dissolved in acetonitrile (15 mL) and water (2.5 mL). Bis(trifluoroacetoxy)iodobenzene (3.94 g, 9.17 mmol) in acetonitrile (10 mL) was slowly added at room temperature to the vigorously stirred solution. After 20 min, TLC analysis (20% EtOAc/hexane) indicated complete reaction. EtOAc (150 mL) was added, and the mixture was rinsed with saturated sodium bicarbonate solution (50 mL) and brine (50 mL). The organic fractions were dried, and the solvent was removed in vacuo. The crude product was purified twice by flash column chromatography (10% EtOAc/hexane) to give 2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3-fluorophenyl)-2-hydroxyethanone (0.853 g, 2.60 mmol, 35%) as a pale yellow oil. Proton NMR was consistent with the proposed structure.

Step 5: Synthesis of 1-(4-(difluoromethoxyh-3-methylphen¡n-2-(3-fluorophenyl)ethane-1,2-d¡one

1-(2,4-Difluorophenyl)-2-(4-methoxy-3-fluorophenyl)ethane-1,2-dione was synthesized according to the representative procedure using 1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-fluorophenyl)-2-hydroxyethanone (0.500 g, 1.612 mmol) to give 1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-fluorophenyl)ethane-1,2-dione (0.3881 g, 78%) as a yellow solid. NMR was consistent with the proposed structure.

Step 6: Synthesis of 2-amino-4-(4-(difluoromethoxhfen¡lM-(3-fluorophen¡n-1-methyl-1H-¡m¡dazol-5(4H)-one

Potassium carbonate (0.516 g, 4.87 mmol) in water (4.6 mL) was added to a mixture of 1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-fluorophenyl)ethane-1,2-dione (0.375 g, 1.217 mmol), 1-methylguanidine hydrochloride (0.533 g, 4.87 mmol), dioxane (19 mL), and ethyl alcohol (25 mL). The reaction mixture was stirred at 85 °C for 4 h. The volatiles were removed in vacuo, and the residue was taken up in chloroform (100 mL) and washed with water (2 x 25 mL). The organic extracts were dried over MgSO4. Evaporation and purification by flash chromatography (60% EtOAc/Hex to 100% EtOAc) followed by recrystallization from cHcb/hexanes gave 2-amino-4-(4-(difluoromethoxy)-3-methylphenyl)-4-(3-fluorophenyl)-1-methyl-1H-imidazol-5(4H)-one (216 mg, 47%) as an off-white solid. 1H NMR (400 MHz, CDCb) 87.37-7.13 (m, 5H), 6.96 (m, 2H), 6.46 (t,J= 74.1 Hz, 1H), 5.73 (s, 2H), 3.09 (s, 3H), 2.23 (s, 3H); 13C NMR (101 MHz, CDCla) 8 178.87, 163.90, 161.46, 155.70, 149.21, 143.36, 138.01, 130.04, 130.02-129.79 (m), 125.73, 122.70 (d,J= 2.9 Hz), 118.75, 116.17, 114.45 (dd,J= 38.7, 22.0 Hz), 113.60, 75.07, 25.87, 16.35. (note: due to the presence of fluorine atoms, J<2c-f>-J<4c-f> couplings are seen giving rise to poorly resolved triplets and doublets); LC (260 nm) Tr (min) = 3.923, LC purity = 96%; m/z:[M+H]+ found = 364.2, [M+H]+ expected = 364.3 (C<17>H<14>F<3>N<3>O).

Example 7 (HCl salt of FAH-17)

2-Amino-4-(4-(Difluoromethoxy)-3-methylphenyl)-4-(3-fluorophenyl)-1-methyl-1H-imidazol-5(4H)-one (0.50 g, 1.38 mmol) was dissolved in anhydrous DCM (66 mL) followed by the addition of HCl (1 M in diethyl ether, 2.2 mL). The mixture was stirred at room temperature for 5 min and the solvent was evaporated in vacuo to give 2-amino-4-(4-(difluoromethoxy)-3-methylphenyl)-4-(3-fluorophenyl)-1-methyl-1H-imidazol-5(4H)-one hydrochloride (0.50 g, 1.20 mmol, 87%) as a white solid. 1H NMR (d6-DMSO): 11.78 (s a, 1H), 9.73 (s a, 1H), 7.53-7.06 (m, 8H), 3.19 (s, 3H), 2.23 (s, 3H); 13C NMR (d6-DMSO): 176.62, 176.99, 172.57, 163.63, 161.20, 157.95, 150.07, 150.04, 140.36, 140.30, 134.40, 129.86, 126.60, 123.70, 119.52, 119.00, 116.96, 116.47, 116.26, 114.64, 114.41, 70.30, 27.43, 16.40 (note: due to the presence of fluorine atoms, J<2c-f>-J<4c-f> couplings are observed, giving rise to triplets and poorly resolved doublets); LC (220 nm): Rt = 3.84 min, 96.5% purity; MS: For C<18>H<16>F<3>N<3>O<2>, [M+H]+ expected = 364.3, obtained 364.1

Example 8 Synthesis of FAH-22

Synthesis of 2-(3-fluoro-5-methylphenyl)-1,3-dithane

BF3.OEt2 (2.61 mL, 21.16 mmol) was added dropwise to a solution of 1,3-propanedithiol (2.48 mL, 24.47 mmol) and 3-fluoro-5-methylbenzaldehyde (3.38 g, 24.47 mmol) in DCM (122 mL) at 0 °C. The reaction was stirred at room temperature for 1 h, where TLC analysis (5% EtOAc/hexane) indicated complete reaction. The reaction mixture was then diluted with DCM (100 mL), filtered through Celite (and the Celite pad was washed with additional DCM (3 x 100 mL)) and the filtrate was washed with brine (100 mL), saturated NaHCO3 (3 x 100 mL), 10% KOH solution (100 mL), water (100 mL), brine (100 mL) and finally dried over sodium sulfate. The organic extract was filtered and evaporated to afford 2-(3-fluoro-5-methylphenyl)-1,3-dithiane (4.69 g, 77%) as a light pink solid. The product was used in the next step without further purification. NMR was consistent with the proposed structure.

Synthesis of 4-(difluoromethoxy)-3-methylbenzaldehyde

A solution of sodium chlorodifluoroacetate (2.60 g, 17.04 mmol) and 4-hydroxy-3-methylbenzaldehyde (1.16 g, 8.52 mmol) in DMF (15 mL) was added over 3 hours to a solution of DMF (15 mL) containing potassium carbonate (1.77 g, 12.78 mmol) at 95 °C. The reaction was allowed to proceed for a further 15 minutes and then cooled. The reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (3 x 50 mL). The organic extract was washed with 10% (m/v) aqueous LiCl solution (3 x 25 mL), dried over sodium sulfate, filtered, and evaporated to give a residue which was subjected to flash chromatography (15% EtOAc/hexane) to give 4-(difluoromethoxy)-3-methylbenzaldehyde (021GLM-053_1(2), 1.00 g, 5.37 mmol, 63%) as a yellow oil. This oil was combined with that from the previous experiment and passed through a Pasteur pipette column eluting with 10% EtOAc/hexane to give an oil that solidified upon standing (1.315 g, 7.06 mmol, 67% for the two reactions). Proton NMR was consistent with the proposed structure. 1.4 g of the desired product was isolated.

Synthesis of (4-(difluoromethox¡)-3-methylphen¡lH2-(3-fluoro-5-methylphen¡l)-1.3-dithan-2-yl)methanol

(4-(Difluoromethoxy)-3-methylphenyl)(2-(3-fluoro-5-methylphenyl)-1,3-dithian-2-yl)methanol was prepared according to the representative procedure using 2-(3-fluoro-5-methylphenyl)-1,3-dithiane (0.932 g, 4.08 mmol) and 4-(difluoromethoxy)-3-methylbenzaldehyde (0.760 g, 4.08 mmol) which gave (4-(difluoromethoxy)-3-methylphenyl)(2-(3-fluoro-5-methylphenyl)-1,3-dithian-2-yl)methanol (0.413 g, 24%) as a yellow oil. NMR was consistent with the proposed structure.

Synthesis of 2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3-fluoro-5-methylphenyl)-2-hydroxyethanone

2-(4-(Difluoromethoxy)-3-methylphenyl)-1-(3-fluoro-5-methylphenyl)-2-hydroxyethanone was synthesized according to the representative procedure using (4-(difluoromethoxy)-3-methylphenyl)(2-(3-fluoro-5-methylphenyl)-1,3-dithian-2-yl)methanol (0.400 g, 0.965 mmol) to give 2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3-fluoro-5-methylphenyl)-2-hydroxyethanone (162 mg, 47%) as a yellow solid. NMR was consistent with the proposed structure. Note: (4-(Difluoromethoxy)-3-methylphenyl)(2-(3-fluoro-5-methylphenyl)-1,3-dithian-2-yl)methanone (104 mg, 22%) was also recovered.

Synthesis of 1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-fluoro-5-methylphenyl)ethane-1.2-dione

1-(4-(Difluoromethoxy)-3-methylphenyl)-2-(3-fluoro-5-methylphenyl)ethane-1,2-dione was synthesized according to the representative procedure using 2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3-fluoro-5-methylphenyl)-2-hydroxyethanone (0.150 g, 0.463 mmol) to give 1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-fluoro-5-methylphenyl)ethane-1,2-dione (0.1134 g, 74%) as a yellow solid. NMR was consistent with the proposed structure.

Synthesis of 2-amino-4-(4-(difluoromethoxy)-3-methyl phenyl)-4-(3-fluoro-5-methyl phenyl)-1-methyl-1H -mdazol-5(4H)-one

Potassium carbonate (0.149 g, 1.407 mmol) in water (2.3 mL) was added to a mixture of 1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-fluorophenyl)ethane-1,2-dione 1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-fluoro-5-methylphenyl)ethane-1,2-dione (0.1134 g, 0.352 mmol), 1-methylguanidine hydrochloride (0.154 g, 1.407 mmol), dioxane (5.46 mL), and ethyl alcohol (7.10 mL). The reaction mixture was stirred at 85 °C for 1.5 h. The volatiles were removed in vacuo, and the residue was taken up in chloroform (50 mL) and washed with water (2 x 15 mL). The organic extracts were dried over MgSO4. Evaporation and purification five times by flash chromatography (1% methanol in ethyl acetate) gave 2-amino-4-(4-(difluoromethoxy)-3-methylphenyl)-4-(3-fluoro-5-methylphenyl)-1-methyl-1H-imidazol-5(4H)-one (85 mg, 75%) as an off-white solid. 1H NMR (400 MHz, CDCb) 87.30 (d,J =2.0 Hz, 1H), 7.28-7.20 (m, 1H), 7.02 (s, 1H), 6.95 (m, 2H), 6.77 (d,J =9.3 Hz, 1H), 6.45 (t,J= 74.1 Hz, 1H), 5.26 (s , 2H), 3.07 (s, 3H), 2.28 (s, 3H), 2.23 (s, 3H). 13C NMR (101 MHz, CDCla) 8 178.52, 163.88, 161.44, 155.75, 149.22 (t,J =2.5 Hz), 141.88 (dd,J =288.6, 7.8 Hz), 138.02, 130.05 (d,J =9.4 Hz), , 123.30 (d,J =2.5 Hz), 118.75 (d,J= 7.5 Hz), 116.21, 115.32 (d,J =21.0 Hz), 113.63, 111.33 (d,J =23.3 Hz), 74.72, 25.83, 21.46 (d,J= 1.8 Hz), 16.33. (note: due to the presence of fluorine atoms, J<2c-f>-J<4c-f> couplings are observed, giving rise to poorly resolved triplets and doublets); LC (220 nm) Tr (min) = 4.007, purity by LC = 98 %; m/z: [M+H]+ found = 378.2, [M+H]+ expected = 378.4 (C19H18F3N302).

Example 9

Synthesis of FAH-23: 2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3-chlorophenyl)-1-methyl-1H-midazole -5(4H)-one

Step 1: Synthesis of 2-(3-chlorophenyl)-1,3-dithane

BF3.0Et2 (2.28 mL, 18.46 mmol) was added dropwise to a solution of 1,3-propanedithiol (2.16 mL, 21.34 mmol) and 3-chlorobenzaldehyde (3.00 g, 21.34 mmol) in DCM (107 mL) at 0 °C. The reaction was stirred at room temperature for 1 h, where TLC analysis (5% EtOAc/hexane) indicated complete reaction. The reaction mixture was then diluted with DCM (50 mL), filtered through Celite (and the Celite pad was washed with additional DCM (3 x 10 mL)) and the filtrate was washed with brine (100 mL), saturated NaHCO3 (3 x 100 mL), 10% KOH solution (100 mL), water (100 mL), brine (100 mL) and finally dried over sodium sulfate. The organic extract was filtered and evaporated to afford 2-(3-chlorophenyl)-1,3-dithiane (4.62 g, 92%) as a colorless solid. The product was used in the next step without further purification.

Step 2: Synthesis of 4-(difluoromethoxy)-3-methylbenzaldehyde

A solution of sodium chlorodifluoroacetate (2.60 g, 17.04 mmol) and 4-hydroxy-3-methylbenzaldehyde (1.16 g, 8.52 mmol) in DMF (15 mL) was added over 3 hours to a solution of DMF (15 mL) containing potassium carbonate (1.77 g, 12.78 mmol) at 95 °C. The reaction was allowed to proceed for a further 15 minutes and then cooled. The reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (3 x 50 mL). The organic extract was washed with 10% (m/v) aqueous LiCl solution (3 x 25 mL), dried over sodium sulfate, filtered, and evaporated to give a residue which was subjected to flash chromatography (15% EtOAc/hexane) to give 4-(difluoromethoxy)-3-methylbenzaldehyde (021GLM-053_1(2), 1.00 g, 5.37 mmol, 63%) as a yellow oil. This oil was combined with that from the previous experiment and passed through a Pasteur pipette column eluting with 10% EtOAc/hexane to give an oil that solidified upon standing (1.315 g, 7.06 mmol, 67% for the two reactions). Proton NMR was consistent with the proposed structure. 1.4 g of the desired product was isolated.

Step 3: Synthesis of (4-(difluoromethoxy)-3-methylphenyl)(2-(3-chlorophenyl)-1,3-dithian-2-yl)methanol

2-(3-Chlorophenyl)-1,3-dithiane (0.92 g, 3.98 mmol) was dissolved in dry THF (20 mL) and cooled to -29 °C. nBuLi (1.6 M, 2.99 mL, 4.78 mmol) was added dropwise under a nitrogen atmosphere and the mixture was stirred for 30 min at -29 °C to give a deep red solution. A solution of 4-(Difluoromethoxy)-3-methylbenzaldehyde (0.74 g, 3.98 mmol) in THF (19.8 mL) was added dropwise and the mixture was allowed to cool to -29 °C and stirred for 15 min, then warmed to room temperature over 1 h and quenched with saturated ammonium chloride solution (7.5 mL) followed by dilution with EtOAc (50 mL). The organic phase was washed with water (2 x 20 mL) and brine (1 x 20 mL) and dried over sodium sulfate. After filtration and concentration, the crude product was purified by flash column chromatography (15% EtOAc/Hexane) to give (2-(3-chlorophenyl)-1,3-dithian-2-yl)(4-(difluoromethoxy)-3-methylphenyl)methanol (0.81 g, 1.94 mmol, 49%) as an oil. NMR was consistent with the proposed structure.

Step 4: Synthesis of 2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3-chlorophenyl)-2-hydroxyethanone

(4-(Difluoromethoxy)-3-methylphenyl)(2-(3-chlorophenyl)-1,3-dithian-2-yl)methanol (3.07 g, 7.34 mmol) was dissolved in acetonitrile (15 mL) and water (2.5 mL). Bis(trifluoroacetoxy)iodobenzene (3.94 g, 9.17 mmol) in acetonitrile (10 mL) was slowly added at room temperature to the vigorously stirred solution. After 20 min, TLC analysis (20% EtOAc/hexane) indicated complete reaction. EtOAc (150 mL) was added, and the mixture was rinsed with saturated sodium bicarbonate solution (50 mL) and brine (50 mL). The organic fractions were dried, and the solvent was removed in vacuo. The crude product was purified twice by flash column chromatography (10% EtOAc/hexane) to give 2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3-chlorophenyl)-2-hydroxyethanone (0.803 g, 2.4 mmol, 32%) as a pale yellow oil. Proton NMR was consistent with the proposed structure.

Step 5: Synthesis of 1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-chlorophenyl)ethane-1,2-dione

(2-(3-Chlorophenyl)-1,3-dithian-2-yl)(4-(difluoromethoxy)-3-methylphenyl)methanol (0.80 g, 1.92 mmol) was dissolved in dichloromethane (24.29 mL) and tert-butanol (5.14 mL, 53.7 mmol) under a nitrogen atmosphere. Dess-Martin periodinane (2.04 g, 4.80 mmol) was added and the reaction was stirred overnight at room temperature. Sodium thiosulfate (5 mL, 1 M) was added and the layers were separated. The organic phase was washed with sodium hydrogen carbonate and the solvent was evaporated. Purification on a prep plate was carried out by centrifugation. in 25% ethyl acetate-hexane gave 1-(3-chlorophenyl)-2-(4-(difluoromethoxy)-3-methylphenyl)ethane-1,2-dione (0.41 g, 1.27 mmol, 66%) as a yellow solid which was used directly in the next step.

Step 6: Synthesis of 2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3-chlorophenyl)-1-methyl-1H-midazole-5( 4H)-one

Potassium carbonate (0.522 g, 4.93 mmol) in water (7.85 mL) was added to a mixture of 1-(3-chlorophenyl)-2-(4-(difluoromethoxy)-3-methylphenyl)ethane-1,2-dione (0.40 g, 1.23 mmol), 1-methylguanidine hydrochloride (0.54 g, 4.93 mmol), dioxane (19.13 mL), and ethyl alcohol (24.87 mL). The reaction mixture was stirred at 85 °C for 1.5 h. The volatiles were removed in vacuo, and the residue was taken up in chloroform (50 mL) and washed with water (2 x 15 mL). The organic extracts were dried over MgSO4. Evaporation and purification three times by PTLC (1% MeOH in EtOAc) and column chromatography (50-90% ethyl acetate:hexane) gave 2-amino-4-(3-chlorophenyl)-4-(4-(difluoromethoxy)-3-methylphenyl)-1-methyl-1H-imidazol-5(4H)-one (0.20 g, 0.52 mmol, 43%) as an off-white solid. 1H NMR (CDCls): 7.48 (s, 1H), 7.30-7.20 (m, 5H), 7.0 (s, 1H), 6.48 (t, 1H,J =74.1 Hz), 4.50 (bs, 2H), 3.12 (s, 3H), 2.26 (s, 3H); 13C NMR (CDCla): 178.45, 155.74, 149.25, 143.22, 137.90, 134.32, 130.05, 127.12, 125.74, 125.36, 118.78, 116.17, 113.6074.73, 25.89, 16.35 (note: due to the presence of fluorine atoms, J2c-f-J4c-f couplings are seen giving rise to poorly resolved triplets and doublets); LC (220 nm): Rt = 3.95 min, 96.6% purity; MS: For C<18>H<16>C F<2>N<3>O<2>, [M+H]+ expected = 380.8, obtained 380.1

Example 10

Synthesis of the compound FAH-27: 2-amino-4-(4-(difluoromethox¡)phen¡l)-4-(3-methylphen¡l)-1-methyl-1H-im dazole-5(4H)-one

Step 1: Synthesis of 2-(3-methylphenyl)-1,3-dithane

BF3.OEt2 (2.67 mL, 21.60 mmol) was added dropwise to a solution of 1,3-propanedithiol (2.53 mL, 24.97 mmol) and 3-methylbenzaldehyde (3.00 g, 24.97 mmol) in DCM (125 mL) at 0 °C. The reaction was stirred at room temperature for 1 h, where TLC analysis (5% EtOAc/hexane) indicated complete reaction. The reaction mixture was then diluted with <d>C<m>(50 mL), filtered through Celite (and the Celite pad was washed with additional DCM (3 x 10 mL)) and the filtrate was washed with brine (100 mL), saturated NaHCO3 (3 x 100 mL), 10% KOH solution (100 mL), water (100 mL), brine (100 mL) and finally dried over sodium sulfate. The organic extract was filtered and evaporated to afford 2-(m-tolyl)-1,3-dithiane (4.66 g, 85%) as a light brown solid. The product was used in the next step without further purification. Proton NMR was consistent with the proposed structure.

Step 2: Synthesis of 4-(difluoromethoxy)-3-methylbenzaldehyde

A solution of sodium chlorodifluoroacetate (2.60 g, 17.04 mmol) and 4-hydroxy-3-methylbenzaldehyde (1.16 g, 8.52 mmol) in DMF (15 mL) was added over 3 hours to a solution of DMF (15 mL) containing potassium carbonate (1.77 g, 12.78 mmol) at 95 °C. The reaction was allowed to proceed for a further 15 minutes and then cooled. The reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (3 x 50 mL). The organic extract was washed with 10% (m/v) aqueous LiCl solution (3 x 25 mL), dried over sodium sulfate, filtered, and evaporated to give a residue which was subjected to flash chromatography (15% EtOAc/hexane) to give 4-(difluoromethoxy)-3-methylbenzaldehyde (021GLM-053_1(2), 1.00 g, 5.37 mmol, 63%) as a yellow oil. This oil was combined with that from the previous experiment and passed through a Pasteur pipette column eluting with 10% EtOAc/hexane to give an oil that solidified upon standing (1.315 g, 7.06 mmol, 67% for the two reactions). Proton NMR was consistent with the proposed structure. 1.4 g of the desired product was isolated.

Stage 3: Synthesis of (4-(difluoromethox¡)-3-met¡lfen¡l)(2-(3-met¡lfen¡l)-1.3-dithane-2-¡l)methanol

2-(m-Tolyl)-1,3-dithiane (2.00 g, 9.51 mmol) was dissolved in dry THF (47.5 mL) and cooled to -29 °C. nBuLi (1.6 M, 7.13 mL, 11.41 mmol) was added dropwise under a nitrogen atmosphere and the mixture was stirred for 30 min at -10 °C to give a deep red solution. A solution of 4-(Difluoromethoxy)-3-methylbenzaldehyde (1.77 g, 9.51 mmol) in THF (47.5 mL) was added dropwise and the mixture was allowed to cool to -29 °C and stirred for 15 min, then warmed to room temperature over 1 h and quenched with saturated ammonium chloride solution (7.5 mL) followed by dilution with EtOAc (50 mL). The organic phase was washed with water (2 x 20 mL) and brine (1 x 20 mL) and dried over sodium sulfate. After filtration and concentration, the crude product was purified by flash column chromatography (15% EtOAc/Hex) to give (4-(difluoromethoxy)-3-methylphenyl)(2-(m-tolyl)-1,3-dithian-2-yl)methanol (2.73 g, 6.88 mmol, 72%) as an oil. NMR was consistent with the proposed structure.

Step 4: Synthesis of 2-(4-(difluoromethoxy¡)-3-met¡lfen¡n-1-(3-met¡lfen¡l)-2-hydroxy¡ethanone

(4-(Difluoromethoxy)-3-methylphenyl)(2-(m-tolyl)-1,3-dithian-2-yl)methanol (2.70 g, 6.81 mmol) was dissolved in dichloromethane (86 mL) and tert-butanol (18.28 mL, 191 mmol) under a nitrogen atmosphere. Dess-Martin periodinane (7.22 g, 17.02 mmol) was added and the reaction was stirred overnight at room temperature. Sodium thiosulfate (5 mL, 1 M) was added and the layers were separated. The organic phase was washed with sodium hydrogen carbonate and the solvent was evaporated. Purification by column chromatography in 5% ethyl acetate/hexane gave 1-(4-(difluoromethoxy)-3-methylphenyl)-2-(m-tolyl)ethane-1,2-dione (1.44 g, 4.75 mmol, 70%) as a yellow solid.

Step 5: Synthesis of 1-(4-(difluoromethoxy)-3-methylphenin-2-(3-methylphenyl)ethane-1,2-d¡one

(4-(Difluoromethoxy)-3-methylphenyl)(2-(m-tolyl)-1,3-dithian-2-yl)methanol (2.70 g, 6.81 mmol) was dissolved in dichloromethane (86 mL) and tert-butanol (18.28 mL, 191 mmol) under a nitrogen atmosphere. Dess-Martin periodinane (7.22 g, 17.02 mmol) was added and the reaction was stirred overnight at room temperature. Sodium thiosulfate (5 mL, 1 M) was added and the layers were separated. The organic phase was washed with sodium hydrogen carbonate and the solvent was evaporated. Purification by column chromatography in 5% ethyl acetate/hexane gave 1-(4-(difluoromethoxy)-3-methylphenyl)-2-(m-tolyl)ethane-1,2-dione (1.44 g, 4.75 mmol, 70%) as a yellow solid and was used directly in the next step.

Step 6: Synthesis of 2-amino-4-(4-(difluoromethox¡)phen¡l)-4-(3-methylphen¡l)-1-methyl-1H-m¡ dazole-5(4H)-one

Potassium carbonate (1.81 g, 17.09 mmol) in water (27.23 mL) was added to a mixture of 1-(4-(difluoromethoxy)-3-methylphenyl)-2-(m-tolyl)ethane-1,2-dione (1.30 g, 4.27 mmol), 1-methylguanidine hydrochloride (1.87 g, 17.09 mmol), dioxane (66.3 mL), and ethyl alcohol (86 mL). The reaction mixture was stirred at 85 °C for 1.5 h. The volatiles were removed in vacuo, and the residue was taken up in chloroform (50 mL) and washed with water (2 x 15 mL). The organic extracts were dried over MgSO4. Evaporation and purification three times by PTLC (1% MeOH in EtOAc) and once by column chromatography (50-90% ethyl acetate:hexane) gave 2-amino-4-(4-(difluoromethoxy)-3-methylphenyl)-1-methyl-4-(m-tolyl)-1H-imidazol-5(4H)-one (0.39 g, 1.07 mmol, 25%) as an off-white solid. 1H NMR (CDCb): 7.34 (s, 1H), 7.24-7.10 (m, 5H), 6.95 (d, 1H,J =8 Hz), 6.47 (t, 1H,J =74.1 Hz), 6.05 (s a, 2H), 3.04 (s, 3H), 2.30 (s, 3H), H); 13C NMR (CDCla): 178.55, 155.97, 149.14, 149.12, 149.09, 141.16, 138.14, 130.20, 128.35, 127.56, 125.94, 124.16, 118.83, 118.61, 116.25, 113.67, 74.74, 25.70, 21.52, 16.32 (note: due to the presence of fluorine atoms, J<2c-f>-J<4c-f> couplings are seen giving rise to poorly resolved triplets and doublets); LC (220 nm): Rt = 3.85 min, 97.3% purity; MS: For C19H19F2N3O2, [M+H]+ expected = 360.4, obtained 360.2

Example 11

Synthesis of FAH-28: 2-amino-4-(4-(difluoromethoxy)phenyl)-4-(3-(trifluoromethyl)phenyl)-1-methyl-1H-midazoline-5(4H)-one

Stage 1: Synthesis of 2-(3-trifluoromethyl)phenyl)-1,3-dithane

BF3.OEt2 (1.84 mL, 14.90 mmol) was added dropwise to a solution of 1,3-propanedithiol (1.74 mL, 17.23 mmol) and 3-(trifluoromethyl)benzaldehyde (3.00 g, 17.23 mmol) in DCM (86 mL) at 0 °C. The reaction was stirred at room temperature for 1 h, where TLC analysis (5% EtOAc/hexane) indicated complete reaction. The reaction mixture was then diluted with DCM (50 mL), filtered through Celite (and the Celite pad was washed with additional DCM (3 x 10 mL)) and the filtrate was washed with brine (100 mL), saturated NaHCO3 (3 x 100 mL), 10% KOH solution (100 mL), water (100 mL), brine (100 mL) and finally dried over sodium sulfate. The organic extract was filtered and evaporated to give 2-(3-(trifluoromethyl)phenyl)-1,3-dithiane (4.62 g, 17.30 mmol, 100%) as a colorless solid. The product was used in the next step without further purification.

Step 2: Synthesis of 4-(difluoromethoxy)-3-methylbenzaldehyde

A solution of sodium chlorodifluoroacetate (2.60 g, 17.04 mmol) and 4-hydroxy-3-methylbenzaldehyde (1.16 g, 8.52 mmol) in DMF (15 mL) was added over 3 hours to a solution of DMF (15 mL) containing potassium carbonate (1.77 g, 12.78 mmol) at 95 °C. The reaction was allowed to proceed for a further 15 minutes and then cooled. The reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (3 x 50 mL). The organic extract was washed with 10% (m/v) aqueous LiCl solution (3 x 25 mL), dried over sodium sulfate, filtered, and evaporated to give a residue which was subjected to flash chromatography (15% EtOAc/hexane) to give 4-(difluoromethoxy)-3-methylbenzaldehyde (021GLM-053_1(2), 1.00 g, 5.37 mmol, 63%) as a yellow oil. This oil was combined with that from the previous experiment and passed through a Pasteur pipette column eluting with 10% EtOAc/hexane to give an oil that solidified upon standing (1.315 g, 7.06 mmol, 67% for the two reactions). Proton NMR was consistent with the proposed structure. 1.4 g of the desired product was isolated.

Stage 3: Synthesis of (4-(difluoromethox¡)-3-methylphen¡l)(2-(3-(trifluoromethox¡n¡l)-1,3-dithane-2-¡ l)methanol

2-(3-(Trifluoromethyl)phenyl)-1,3-dithiane (2.00 g, 7.57 mmol) (021 STM-080) was dissolved in dry THF (38 mL) and cooled to -29 °C. nBuLi (1.6 M, 5.67 mL, 9.08 mmol) was added dropwise under a nitrogen atmosphere and the mixture was stirred for 30 min at -29 °C to give a deep red solution. A solution of 4-(Difluoromethoxy)-3-methylbenzaldehyde (1.41 g, 7.57 mmol) in THF (38 mL) was added dropwise and the mixture was allowed to cool to -29 °C and stirred for 15 min, then warmed to room temperature over 1 h and quenched with saturated ammonium chloride solution (7.5 mL) followed by dilution with EtOAc (50 mL). The organic phase was washed with water (2 x 20 mL) and brine (1 x 20 mL) and dried over sodium sulfate. After filtration and concentration, the crude product was purified by flash column chromatography (5-15% EtOAc/Hex) to give (4-(difluoromethoxy)-3-methylphenyl)(2-(3-(trifluoromethyl)phenyl)-1,3-dithian-2-yl)methanol (2.29 g, 4.55 mmol, 60%) as a golden oil.

Step 4: Synthesis of 2-(4-(difluoromethoxy)-3-methylphenyl)-1-(3-(trifluoromethyl)phenyl)-2-hydroxyethanone

(4-(Difluoromethoxy)-3-methylphenyl)(2-(3-trifluoromethylphenyl)-1,3-dithian-2-yl)methanol (3.07 g, 7.34 mmol) was dissolved in acetonitrile (15 mL) and water (2.5 mL). Bis(trifluoroacetoxy)iodobenzene (3.94 g, 9.17 mmol) in acetonitrile (10 mL) was slowly added at room temperature to the vigorously stirred solution. After 20 minutes, TLC analysis (20% EtOAc/hexane) indicated complete reaction. EtOAc (150 mL) was added, and the mixture was rinsed with saturated sodium bicarbonate solution (50 mL) and brine (50 mL). The organic fractions were dried, and the solvent was removed in vacuo. The crude product was purified twice by flash column chromatography (10% EtOAc/hexane) to give 2-(4-(difluoromethoxy)-3-trifluoromethylphenyl)-1-(3-methylphenyl)-2-hydroxyethanone (0.803 g, 2.4 mmol, 32%) as a pale yellow oil. Proton NMR was consistent with the proposed structure.

Step 5: Synthesis of 1-(4-(difluoromethoxy)-3-(trifluoromethyl)phenoxy)-2-(3-(trifluoromethylphenoxy)ethane-1,2-doxy)

(4-(Difluoromethoxy)-3-methylphenyl)(2-(3-(trifluoromethyl)phenyl)-1,3-dithian-2-yl)methanol (2.20 g, 4.88 mmol) was dissolved in dichloromethane (61.8 mL) and tert-butanol (13.08 mL, 137 mmol) under a nitrogen atmosphere. Dess-Martin periodinane (5.18 g, 12.21 mmol) was added and the reaction was stirred overnight at room temperature. Sodium thiosulfate (5 mL, 1 M) was added and the layers were separated. The organic phase was washed with sodium hydrogen carbonate and the solvent was evaporated. Purification by column chromatography in 5% ethyl acetate/hexane gave 1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-(trifluoromethyl)phenyl)ethane-1,2-dione (1.20 g, 3.35 mmol, 69%) as a yellow solid which was used directly in the next step.

Potassium carbonate (1.36 g, 12.84 mmol) in water (20.46 mL) was added to a mixture of 1-(4-(difluoromethoxy)-3-methylphenyl)-2-(3-(trifluoromethyl)phenyl)ethane-1,2-dione (1.15 g, 3.21 mmol), 1-methylguanidine hydrochloride (1.41 g, 12.84 mmol), dioxane (49.8 mL), and ethyl alcohol (64.8 mL). The reaction mixture was stirred at 85 °C for 1.5 h. The volatiles were removed in vacuo, and the residue was taken up in chloroform (50 mL) and washed with water (2 x 15 mL). The organic extracts were dried over MgSO4. Evaporation and purification three times by PTLC (1% MeOH in EtOAc) and once by column chromatography (50-90% ethyl acetate:hexane) gave 2-amino-4-(3,5-difluorophenyl)-4-(6-methoxypyridin-3-yl)-1-methyl-1H-imidazol-5(4H)-one (0.18 g, 0.44 mmol, 14%) as an off-white solid. 1H NMR (CDCb): 7.80 (s, 1H), 7.70 (d, 1H, J = 8 Hz), 7.55 (d, 1H, J = 8 Hz), 7.45-7.41 (m, 1H), 7.32 (s, 1H), 7.28-7.24 (m, 1H), 7.0 (d, 1H,J =8 Hz), 48 (t, 1H,J =74.1 Hz), 5.64 (s a, 2H), 3.10 (s, 3H), 2.26 (s, 3H); 13C NMR (CDCla): 178.78, 156.18, 156.09, 156.07, 149.23, 142.39, 138.11, 130.71, 130.13, 128.99, 125.72, 122.72, 118.73, 116.15, 113.57, 75.05, 25.84, 16.33 (note: due to the presence of fluorine atoms, J2c-f-J4c-f couplings are seen giving rise to poorly resolved triplets and doublets); LC (220 nm): Rt = 4.04 min, 96.6% purity; MS: For C19H16F5N3O2, [M+H]+ expected = 414.3, obtained 414.1

Example 12

Analysis by SPR

The surfaces of the two flow cells FC1 and FC2 of a carboxymethylated dextran chip (CM-5) were washed sequentially with 50 mM NaOH, 1 mM HCl, 0.05% H3PO4 and 20 mM sodium phosphate pH 7.4, 125 mM sodium chloride in parallel using a flow rate of 30 µl/min for 1 minute using a Biacore T-100 (GE Healthcare). The fusion protein was immobilized by amine coupling using 20 mM phosphate, 125 mM sodium chloride pH 7.4 onto FC2. This TRX-eAPP575-624 fusion protein contains a fusion of thioredoxin (TRX) and residues 575-624 of the APP ectodomain (20 kDa). The fusion protein is produced as described in Libeue et al.(JAD 2011). The protein was concentrated to 2 mg/ml in 20 mM phosphate at pH 6.5, 125 mM sodium chloride and then dissolved to a concentration of 50 pg per ml in 20 mM sodium acetate at pH 5.0. FC1 served as a reference cell after mock immobilization with buffer alone. For all cells, the flow rate was 10 µl per minute. The chip was blocked with 1 M ethanolamine (pH 8.5). Final RU values were determined for BACE inhibitors binding to TRX-eAPP575-624 by flowing varying concentrations of the inhibitor into DMSO at 50 pM. Compounds were diluted from 10 mM solutions in DMSO to 50 pM in 1% DMSO, 20 mM sodium phosphate pH 7.4, 125 mM sodium chloride, 0.05% Tween and then serially diluted 1.5-fold over 10 steps. Binding traces were recorded for each dilution with a 60 s binding phase and a 240 s dissociation phase. Each cycle was run at 20 °C with a constant flow rate of 20 µl/min. An additional 240 s of buffer flow at 60 µl per minute was applied through the cells as a regeneration phase to facilitate complete dissociation of the compound from the protein. Sensorgrams were obtained by subtracting the reference and buffer signals using Biacore T100 evaluation software. Binding curves were modeled with PRISM (Graphpad Inc).

Example 13

Experimental methods for measuring AB42 in SH-SY5Y cells

Abeta in vitro test assay:

SH-SY5Y neuroblastoma cells were seeded at 50,000 cells/well in a 96-well plate for 24 h. Their medium was then changed with fresh medium supplemented with the desired concentration of the hydantoin compound (e.g., compound 3). After 24 h, 20 µl of the medium was added to 2 µl of the complete protease inhibitor with 1 µM EDTA and kept at 4 °C until analysis by the ELISA assay below.

ELISA assays:

Invitrogen ELISA kits were also used to quantitate Ap1-42 (KHB3544) in duplicate. For the ultrasensitive Ap 1-42 ELISA, samples were diluted 1:2 (50 µl CSF plus 50 µl standard diluent buffer provided by the kit). Assays were performed according to the manufacturer's instructions. Briefly, standards and samples were added to a plate precoated with a monoclonal capture antibody specific for the amino terminus of Hu Ap. Samples were co-incubated with a rabbit detection antibody (Ab) specific for the carboxy terminus of the Ap species that was assayed for 3 h at room temperature overnight at 4° (Ap 1-42) with gentle rocking. After washing, bound rabbit Ab was detected using a horseradish peroxidase-labeled anti-rabbit secondary Ab. After washing again, bound HRP-anti-rabbit Ab (Spectramax 190, Molecular Devices) was detected colorimetrically by the addition of substrate solution. 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) protease inhibitor (101500, Calbiochem) was added to the standards and samples.

Example 14

Brain uptake test (PK)

In general, the exposure of hydantoins to the CNS was carried out as follows:

The studies consisted of the extraction of plasma and heparinized brains after treatment with hydantoins, following subcutaneous (sc) administration of the molecules at a rate of 10 mg/kg. Plasma and brain levels of the compounds were determined by quantitative LC/MS/MS methodology, performed at Integrated Analytical Solutions (online at ianalytical.net). Plasma samples were precipitated with an acetonitrile:methanol (1:1) cocktail containing an internal standard. Brain samples were homogenized directly in ethyl acetate or extracted from 5 M guanidine homogenates by the liquid-liquid method. The resulting supernatant was evaporated to dryness and subjected to LC/MS/MS analysis. For each compound, 3 mice were used for analysis. Brain-plasma ratios and brain levels were then calculated.

Example 15

ABBI Selectivity: Inhibition of APP-BACE versus PSLG1-BACE or NRG1-BACE cleavage

P5-P5' test:

To determine the IC<50>of APP-BACE1, the BACE1 substrate from Sigma (7-methoxycoumarin-4-acetyl-[Asn670, Lue671]-amyloid b/A4, precursor protein 770, fragment 667-676-(2,4-dinitrophenyl)Lys-Arg-Arg trifluoroacetate salt) was used and the manufacturer's protocol was followed. Briefly, 0.01 units of BACE1 was incubated for 1 h at room temperature with a BACE inhibitor, then the substrate was added to each well and fluorescence was read immediately and every 30 min for 2 h. Activity was determined by dividing the fluorescence at a specific [BACE inhibitor] by the fluorescence at [BACE inhibitor] = 0 jM, % activity was plotted against the log of [BACE inhibitor] to determine APP-BACE1 using GraphPad Prism 5 (Figure 6A).

PSGL1 and NRG1 assays:

Briefly, to determine the IC<50>of PSGL-1-BACE1, HEK 293 cells were seeded in 24-well plates and transiently co-transfected with PSGL1/lacZ or NRG1/lacZ constructs using Lipofectamine 2000; and the manufacturer's protocol was followed. Two hours after adding the DNA-lipid complex to the cells, a BACE inhibitor was added to each well, then the cells were incubated overnight at 37°C and 5% CO<2>. The cultured medium was collected to determine NRG1 or PSGL1, and the cells were lysed to measure lacZ levels. The standard Sigma SEAP kit protocol was performed on the cultured medium to detect PSGL1 or NRG1 levels. Promega kit instructions were followed to determine lacZ concentration. The ratio of PSGL1/lacz versus [BACE inhibitor] was plotted to determine the IC<50> of BACE1 on each of the substrates using GraphPad Prism 5 (Figure 6B). ABBI FAH17 shows >200-fold selectivity for APP over PSGL1. Similar tests show that FAH17 is >10-fold selective for APP over NRG1.

Claims (14)

CLAIMS 1. A compound selected from the group consisting of or a pharmaceutically acceptable salt thereof, a tautomer thereof, a pharmaceutically acceptable salt of a tautomer thereof, an enantiomer thereof, or a pharmaceutically acceptable salt of an enantiomer thereof. 2. The compound of claim 1, wherein said compound is capable of binding to APP and/or the BACE enzyme and/or an APP/BACE complex; or wherein said compound is capable of binding to APP and inhibiting the BACE enzyme. 3. A kit comprising one or more containers containing a compound of claim 1 or claim 2. 4. A pharmaceutical formulation comprising a pharmaceutically acceptable carrier and a compound of any one of claims 1 to 3. 5. The formulation of claim 4, wherein said formulation is composed for administration via a route selected from the group consisting of oral delivery, isophoretic delivery, transdermal delivery, parenteral delivery, aerosol administration, administration via inhalation, intravenous administration and rectal administration, preferably, wherein said formulation is composed for oral administration. 6. The formulation of claim 4 or 5, wherein said formulation is sterile and/or said formulation is a unitary pharmaceutical formulation. 7. The compound according to any one of claims 1 to 2, or the formulation of any one of claims 4 to 6 for use in therapy. 8. A hydantoin for use in a method of ameliorating or treating one or more symptoms of a pre-Alzheimer's cognitive condition and/or dysfunction, or delaying the progression of a pre-Alzheimer's cognitive condition or dysfunction to Alzheimer's disease, said method comprising: administering to a subject in need thereof the hydantoin in an amount sufficient to prevent or delay the onset of a pre-Alzheimer's cognitive dysfunction, and/or to ameliorate one or more symptoms of a pre-Alzheimer's cognitive dysfunction, and/or to prevent or delay the progression of a pre-Alzheimer's cognitive dysfunction to Alzheimer's disease, wherein the hydantoin is a compound having a formula selected from the group consisting of (FAH-28), or a pharmaceutically acceptable salt thereof. 9. The hydantoin for the use of claim 8, wherein: said administration results in a reduction in the CSF levels of one or more components selected from the group consisting of Ap42, sAPPp, total Tau (tTau), phospho-Tau (pTau), APPneo, soluble Ap40, pTau/Ap42 ratio, and tTau/Ap42 ratio, and/or the increase in the CSF levels of one or more components selected from the group consisting of Ap42/Ap40 ratio, Ap42/Ap38 ratio, sAPPa, sAPPa/sAPPp ratio, sAPPa/Ap40 ratio, and sAPPa/Ap42 ratio; and/or said administration results in a reduction in the plaque burden in the brain of the subject; and/or said administration results in a reduction in the rate of plaque formation in the brain of the subject; and/or such administration produces an improvement in the subject's cognitive abilities; and/or such administration produces an improvement in, a stabilization of, or a reduction in the rate of decline in the subject's clinical dementia rating (CDR). 10. A hydantoin for use in a method of ameliorating or treating one or more symptoms of Alzheimer's disease, and/or reversing Alzheimer's disease, and/or reducing the rate of progression of Alzheimer's disease, said method comprising: administering to a subject in need thereof the hydantoin in an amount sufficient to ameliorate one or more symptoms of Alzheimer's disease, and/or to reverse Alzheimer's disease, and/or to reduce the rate of progression of Alzheimer's disease, wherein the hydantoin is a compound having a formula selected from the group consisting of: �� and a pharmaceutically acceptable salt thereof. 11. The hydantoin for the use of claim 10, wherein: said subject is diagnosed with early stage Alzheimer's disease; or said subject is diagnosed with mid-stage Alzheimer's disease; or said subject is diagnosed with late-stage Alzheimer's disease; or said subject exhibits a clinical classification of dementia indicative of Alzheimer's disease; and/or said subject has a familial risk for Alzheimer's disease; and/or said subject has a familial Alzheimer's disease (FAD) mutation. 12. The hydantoin for the use of claim 10 or 11, wherein: said administration improves one or more symptoms of Alzheimer's disease; and/or said administration reduces the rate of progression of Alzheimer's disease; and/or said administration results in a reduction in the CSF of the levels of one or more components selected from the group consisting of Ap42, sAPPp, total Tau (tTau), phospho-Tau (pTau), APPneo, soluble Ap40, pTau/Ap42 ratio, and tTau/Ap42 ratio, and/or the increase in the CSF of the levels of one or more components selected from the group consisting of the Ap42/Ap40 ratio, the Ap42/Ap38 ratio, sAPPa, the sAPPa/sAPPp ratio, the sAPPa/Ap40 ratio, and the sAPPa/Ap42 ratio; and/or said administration results in a reduction of plaque load in the subject's brain; and/or said administration results in a reduction in the rate of plaque formation in the subject's brain; and/or said administration results in an improvement in the subject's cognitive abilities; and/or said administration results in an improvement in, a stabilization of, or a reduction in the rate of decline in the subject's clinical dementia rating (CDR); and/or said administration results in a perceived improvement in the subject's quality of life; and/or said administration results in reduced cerebral amyloidosis and/or downstream neurodegeneration. 13. A hydantoin for use in a method of treatment that slows the progression, stops or reverses age-related macular degeneration (AMD) in a mammal, said method comprising administering to said mammal the hydantoin in an amount sufficient to slow the progression, stop or reverse age-related macular degeneration in said mammal, wherein the hydantoin is a compound having a formula selected from the group consisting of: a pharmaceutically acceptable salt thereof. 14. A hydantoin for use in the treatment of Alzheimer's disease, cognitive impairment, Down syndrome, HCHWA-D, cognitive impairment, senile dementia, cerebral amyloid angiopathy, or a neurodegenerative disorder, wherein the hydantoin is a compound having a formula selected from the group consisting of (FAH-28), or a pharmaceutically acceptable salt thereof.